Phosphoinositide 3-kinase inhibitor with a zinc binding moiety

ABSTRACT

The invention provides a compound of Formula I, 
     
       
         
         
             
             
         
       
     
     Pharmaceutical compositions comprising such compounds and the use of such compounds in the treatment of phosphoinositide 3-kinase related diseases and disorders such as cancer. The instant application further relates to the the treatment of histone deacetylase related disorders and diseases related to both histone deacetylase and phosphoinositide 3-kinase.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/197,769, filed Mar. 5, 2014 which is a continuation of U.S.application Ser. No. 13/435,062, filed on Mar. 30, 2012, now U.S. Pat.No. 8,710,219, issued Apr. 29, 2014, which claims the benefit of U.S.Provisional Application No. 61/470,849, filed on Apr. 1, 2011 and61/559,489, filed on Nov. 14, 2011. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Phosphoinositides (PIs), which are phosphorylated derivatives ofphosphatidylinositol, are essential in eukaryotic cells, regulatingnuclear processes, cytoskeletal dynamics, signalling and membranetrafficking. Among the enzymes involved in PI metabolism, PI3-kinases(PI3K) have attracted special attention because of their oncogenicproperties and potential as drug targets. PI3-kinases phosphorylatephosphatidylinositols or PIs at the 3-position of the inositol ring.(Lindmo et al. Journal of Cell Science 119, 605-614, 2006). The3-phosphorylated phospholipids generated by PI3K activity bind to thepleckstrin homology (PH) domain of protein kinase B (PKB), causingtranslocation of PKB to the cell membrane and subsequent phosphorylationof PKB. Phosphorylated PKB inhibits apoptosis-inducing proteins such asFKHR, Bad, and caspases, and is thought to play an important role incancer progression. The PI3Ks are divided into classes and class I isfurther subclassified into classes Ia and Ib. Among these isoforms,class Ia enzymes are thought to play the most important role in cellproliferation in response to growth factor-tyrosine kinase pathwayactivation (Hayakawa et al., Bioorganic & Medicinal Chemistry 146847-6858, 2006). Three frequent mutations in cancer constitutivelyactivate PI3Ka and, when expressed in cells, they drive the oncogenictransformation and chronic activation of downstream signalling bymolecules such as PKB, S6K and 4E bp1 that is commonly seen in cancercells. (Stephens et al., Current Opinion in Pharmacology, 5(4) 357-365,2005). As such, PI3-kinases are attractive targets for the treatment ofproliferative diseases.

There are several known PI3-kinase inhibitors including Wortmannin andLY294002. Although Wortmannin is a potent PI3K inhibitor with a lownanomolar IC₅₀ value, it has low in vivo anti-tumor activity. (Hayakawaet al., Bioorg. Med. Chem. 14(20), 6847-6858 (2006)). Recently, a groupof morpholine substituted quinazoline, pyridopyrimidine andthienopyrimidine compounds have been reported to be effective ininhibiting PI3kinase p110α. (Hayakawa, 6847-6858). Oral dosage of amorpholine substituted thienopyrimidine compound (GDC-0941) has showntumor suppression in glioblastoma xenografts in vivo. (Folkes et al.,Journal of Medicinal Chemistry, 51, 5522-5532, 2008). The followingpublications disclose a series of thienopyrimidine, pyridopyrimidine andquinazoline based PI3-Kinase inhibitors: WO 2008/073785; WO 2008/070740;WO 2007/127183; U.S. Patent Publication 20080242665.

Histone acetylation is a reversible modification, with deacetylationbeing catalyzed by a family of enzymes termed histone deacetylases(HDACs). HDAC's are represented by 18 genes in humans and are dividedinto four distinct classes (J Mol Biol, 2004, 338:1, 17-31). Inmammalians class I HDAC's (HDAC1-3, and HDAC8) are related to yeast RPD3HDAC, class 2 (HDAC4-7, HDAC9 and HDAC10) related to yeast HDA1, class 4(HDAC11), and class 3 (a distinct class encompassing the sirtuins whichare related to yeast Sir2).

Csordas, Biochem. J., 1990, 286: 23-38 teaches that histones are subjectto post-translational acetylation of the ε-amino groups of N-terminallysine residues, a reaction that is catalyzed by histone acetyltransferase (HAT1). Acetylation neutralizes the positive charge of thelysine side chain, and is thought to impact chromatin structure. Indeed,access of transcription factors to chromatin templates is enhanced byhistone hyperacetylation, and enrichment in underacetylated histone H4has been found in transcriptionally silent regions of the genome(Taunton et al., Science, 1996, 272:408-411). In the case of tumorsuppressor genes, transcriptional silencing due to histone modificationcan lead to oncogenic transformation and cancer.

Several classes of HDAC inhibitors currently are being evaluated byclinical investigators. Examples include hydroxamic acid derivatives,Suberoylanilide hydroxamic acid (SAHA), PXD101 and LAQ824, are currentlyin the clinical development. In the benzamide class of HDAC inhibitors,MS-275, MGCD0103 and CI-994 have reached clinical trials. Mourne et al.(Abstract #4725, AACR 2005), demonstrate that thiophenyl modification ofbenzamides significantly enhance HDAC inhibitory activity against HDAC1.

Certain cancers have been effectively treated with such a combinatorialapproach; however, treatment regimes using a cocktail of cytotoxic drugsoften are limited by dose limiting toxicities and drug-druginteractions. More recent advances with molecularly targeted drugs haveprovided new approaches to combination treatment for cancer, allowingmultiple targeted agents to be used simultaneously, or combining thesenew therapies with standard chemotherapeutics or radiation to improveoutcome without reaching dose limiting toxicities. However, the abilityto use such combinations currently is limited to drugs that showcompatible pharmacologic and pharmacodynamic properties. In addition,the regulatory requirements to demonstrate safety and efficacy ofcombination therapies can be more costly and lengthy than correspondingsingle agent trials. Once approved, combination strategies may also beassociated with increased costs to patients, as well as decreasedpatient compliance owing to the more intricate dosing paradigmsrequired.

SUMMARY OF THE INVENTION

The present invention relates to a compound of Formula I:

and pharmaceutically acceptable salts thereof, where R is hydrogen or anacyl group. The acyl group is preferably R₁C(O)—, where R₁ issubstituted or unsubstituted C₁-C₂₄-alkyl, preferably C₁-C₁₀-alkyl, andmore preferably C₁-C₆-alkyl; substituted or unsubstitutedC₂-C₂₄-alkenyl, preferably C₂-C₁₀-alkenyl, and more preferablyC₂-C₆-alkenyl; substituted or unsubstituted C₂-C₂₄-alkynyl, preferablyC₂-C₁₀-alkynyl, and more preferably C₂-C₆-alkynyl; substituted orunsubstituted aryl, preferably substituted or unsubstituted phenyl; orsubstituted or unsubstituted heteroaryl.

The invention also relates to pharmaceutical compositions comprising acompound of Formula I, or a pharmaceutically acceptable salt thereof, incombination with a pharmaceutically acceptable excipient or carrier.

The compounds of Formula I and, in particular, Compound 1, haveadvantageous properties for use as therapeutic agents, such as for thetreatment of cancers and other diseases and disorders associated withPI3 kinase actitivy and/or HDAC activity. Compound 1, for example, haspotent inhibitory activity against the molecular targets PI3K and HDACand potent antiproliferative activity against a variety of cancer celllines in vitro. Compound 1 has significant oral bioavailability asobserved in animal models. Upon either oral or intravenous dosing inxenograft tumor bearing mice, the compound shows significant uptake bythe tumor tissue and pharmacodynamic activity in tumor tissue. Compound1 also shows substantial antitumor activity in mouse xenograft tumormodels following either oral or intravenous administration. The compoundalso has a favorable safety profile, as shown, for example, bygenotoxicity testing using the Ames test.

The invention further relates to the use of the compounds of theinvention in the treatment of PI3K related diseases and disorders suchas cancer. These compounds further act as an HDAC inhibitor by virtue ofits ability to bind zinc ions. The compounds are active at multipletherapeutic targets and are effective for treating a variety ofdiseases. Moreover, in some cases it has been found that these compoundshave enhanced activity when compared to the activities of combinationsof separate molecules individually having PI3-Kinase inhibitory activityand HDAC inhibitory activity. In other words, the combination ofPI3-kinase and HDAC inhibitory activity in a single molecule may providea synergistic effect as compared to the PI3-kinase and HDAC inhibitorsseparately.

Moreover, the efficacy of single-agent PI3K pathway inhibitors islimited by the presence of primary/acquired genetic alterations andactivation of multiple pro-survival and growth pathways (Engelman (2009)Nature Reviews Cancer, 9: 550-562). Inhibition of PI3K by single-agentPI3K pathway inhibitors can actually upregulate signaling of theRAF-MEK-ERK pathway by the release of negative feedback loops. Thecompounds of the invention, by virtue of their integrated PI3K/HDACinhibitory activities, provide the potential to overcome the limitationsin the treatment of cancers with single-target PI3K inhibitors. Thecompounds of the invention disrupt cancer networks in in vivo and invitro experiments, resulting from durable inhibition of thePI3K-AKT-mTOR pathway, the inhibition of the RAF-MEK-ERK pathway, andthe downregulation of receptor tyrosine kinase (RTK) protein levels. Inaddition, the compounds of the invention induce cell cycle arrest andapoptosis resulting from the upregulation of tumor suppressors p53 andp21 in tumor cell lines in vitro. Accordingly, compounds of theinvention have the potential to overcome primary and acquired drugresistance and may be more efficacious than mono-treatment withsingle-agent PI3K pathway inhibitors in clinical applications.

Another aspect of the invention provides methods of inhibiting PI3kinase activity, by contacting a PI3 kinase with an effective inhibitoryamount of a compound of Formula I, or a pharmaceutically acceptable saltthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 is a graph of concentration of Compound 1 versus time in plasmaand tumor tissue following oral administration to H2122 xenografttumor-bearing nude mice.

FIG. 2A is a graph of Compound 1 plasma concentration versus time inDaudi xenograft tumor-bearing Scid mice following oral dosing at 25, 50and 100 mg/kg.

FIG. 2B is a graph of Compound 1 tumor concentration versus time inDaudi xenograft tumor-bearing Scid mice following oral dosing at 25, 50and 100 mg/kg.

FIG. 2C is a graph of Compound 1 concentration versus time in plasma andtumor tissue in Daudi xenograft tumor-bearing Scid mice following oraldosing at 100 mg/kg.

FIG. 3 presents Western blots of tumor tissue extracts from control andCompound 1 treated (25, 50 and 100 mg/Kg) Scid mice bearing Daudi tumorxenografts.

FIG. 4 is a graph of Compound 1 plasma concentration versus time inbeagle dogs following oral or intravenous dosing.

FIG. 5A is a graph of tumor growth versus time in H2122 xenografttumor-bearing nude mice treated with Compound 1 or vehicle.

FIG. 5B is a graph of tumor growth versus time in Daudi xenografttumor-bearing nude mice treated with Compound 1 or vehicle.

FIG. 5C is a graph of tumor growth versus time in OPM2 xenografttumor-bearing nude mice treated with Compound 1 or vehicle.

FIG. 6 is a graph showing circulating blood levels of T and Blymphocytes

following treatment with Compound 1 or vehicle.

FIGS. 7A to 7G present Western blots of extracts from control andCompound 1 treated H460 (Kras, PI3K) cells. GDC is GDC-0941; LBH isLBH-589.

FIGS. 8A to 8C present Western blots of extracts from control andCompound 1 treated H1975 (EGFR, PI3K), BT474 (HER2, PI3K), H1975 (EGFR,PI3K), A375 (B-Raf) and RPMI-822 (p53⁻) cells.

FIG. 9 is a graph of tumor growth versus time in Daudi xenografttumor-bearing Scid mice treated orally with Compound 1 or vehicle.

FIG. 10 is a graph of tumor growth versus time in Daudi xenografttumor-bearing Scid mice treated with vehicle, Compound 1, SAH, GDC-0941or a combination of SAHA and GDC-0941.

FIG. 11 is a graph of tumor growth versus time in SU-DHL4 xenografttumor-bearing nude mice treated orally with Compound 1 or vehicle.

FIG. 12 is a graph of tumor growth versus time in OPM2 xenografttumor-bearing nude mice treated with Compound 1 or vehicle.

FIG. 13 is a graph of tumor growth versus time in MM1S xenografttumor-bearing SCID mice treated with Compound 1 or vehicle.

FIG. 14 is a graph of tumor growth versus time in MM1R xenografttumor-bearing SCID mice treated with Compound 1 or vehicle.

FIG. 15 presents Western blots of tumor extracts from Compound 1 treatedSCID mice bearing Daudi, SuDHL-4, HS-Sultan, DOHH-2, OPM-2, MM1R or MM1Sxenograft tumors.

FIG. 16 is a graph of tumor growth versus time in Daudi tumor-bearingSCID mice treated with Compound 1, CAL-101 or vehicle.

FIG. 17 is a graph of tumor growth versus time in Daudi tumor-bearingSCID mice treated with Compound 1, cyclophosphamide, combination ofCompound 1 and cyclophosphamide or vehicle.

FIG. 18 is a graph of tumor growth versus time in MM1S tumor-bearingSCID mice treated with Compound 1, lenalidomide, combination of Compound1 and lenalidomide or vehicle.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment, the compound of Formula I is set forth below:

(hereinafter “Compound 1”, also referred to asN-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamideor a pharmaceutically acceptable salt thereof.

The invention further provides methods for the prevention or treatmentof diseases or conditions involving aberrant proliferation,differentiation or survival of cells. In one embodiment, the inventionfurther provides for the use of one or more compounds of the inventionin the manufacture of a medicament for halting or decreasing diseasesinvolving aberrant proliferation, differentiation, or survival of cells.In a preferred embodiment, the disease is cancer. In one embodiment, theinvention relates to a method of treating cancer in a subject in need oftreatment comprising administering to said subject a therapeuticallyeffective amount of a compound of the invention.

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemias, lymphomas and the like. For example, cancersinclude, but are not limited to, mesothelioma, leukemias and lymphomassuch as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheralT-cell lymphomas, lymphomas associated with human T-cell lymphotrophicvirus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-celllymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia,chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, andmultiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL),chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma,adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronicmyeloid leukemia (CIVIL), or hepatocellular carcinoma. Further examplesinclude myelodisplastic syndrome, childhood solid tumors such as braintumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal),genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian,testicular), lung cancer (e.g., small-cell and non small cell), breastcancer, pancreatic cancer, melanoma and other skin cancers, stomachcancer, brain tumors, tumors related to Gorlin's syndrome (e.g.,medulloblastoma, meningioma, etc.), and liver cancer. Additionalexemplary forms of cancer which may be treated by the subject compoundsinclude, but are not limited to, cancer of skeletal or smooth muscle,stomach cancer, cancer of the small intestine, rectum carcinoma, cancerof the salivary gland, endometrial cancer, adrenal cancer, anal cancer,rectal cancer, parathyroid cancer, and pituitary cancer.

Additional cancers that the compounds described herein may be useful inpreventing, treating and studying are, for example, colon carcinoma,familiary adenomatous polyposis carcinoma and hereditary non-polyposiscolorectal cancer, or melanoma. Further, cancers include, but are notlimited to, labial carcinoma, larynx carcinoma, hypopharynx carcinoma,tongue carcinoma, salivary gland carcinoma, gastric carcinoma,adenocarcinoma, thyroid cancer (medullary and papillary thyroidcarcinoma), renal carcinoma, kidney parenchyma carcinoma, cervixcarcinoma, uterine corpus carcinoma, endometrium carcinoma, chorioncarcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumorssuch as glioblastoma, astrocytoma, meningioma, medulloblastoma andperipheral neuroectodermal tumors, gall bladder carcinoma, bronchialcarcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma,choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma,osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma,Ewing sarcoma, and plasmocytoma. In one aspect of the invention, thepresent invention provides for the use of one or more compounds of theinvention in the manufacture of a medicament for the treatment ofcancer.

In one embodiment, the compounds of the invention are used to treat ahematological cancer or hematological precancerous condition.Hematological cancers include leukemias, lymphomas and multiple myeloma.Examples include lymphocytic leukemias, such as acute lymphocyticleukemia, including precursor B acute lymphoblastic leukemia, precursorT acute lymphoblastic leukemia, Burkitt's leukemia, and acutebiphenotypic leukemia; and chronic lymphocytic leukemia, includingB-cell prolymphocytic leukemia; and myologenous leukemias, such as acutemyologenous leukemia, including acute promyelocytic leukemia, acutemyeloblastic leukemia, and acute megakaryoblastic leukemia; and chronicmyologenous leukemia, including chronic monocytic leukemia; acutemonocytic leukemia. Other leukemias include hairy cell leukemia; T-cellprolymphocytic leukemia; large granular lymphocytic leukemia; and AdultT-cell leukemia. Lymphomas include Hodgkin's lymphoma and Non-Hodgkin'slymphoma, including B-cell lymphomas, T-cell lymphomas, such ascutaneous T-cell lymphoma, and NK cell lymphomas. Hematologicalprecancerous conditions include myelodysplastic syndrome andmyeloproliferative disorders, such as primary myelofibrosis,polycythemia vera, and essential thrombocythemia.

Compounds of the invention have been shown to induce reversiblelymphopenia and are therefore of use for removing or decreasing thecirculating levels of cancer cells of lymphocytic lineage. Suchcompounds are also of use for treating autoimmune disorders or formodulating an immune response.

In one embodiment, the invention provides a method for reducing thecirculating lymphocyte count in a subject, comprising administering tothe subject an effective amount of a compound of the invention. In apreferred embodiment, the reduced circulating lymphocyte count isreversible, that is, the circulating lymphocyte count returns to thenormal range after dosing with the compound of the invention is stopped.In one embodiment, the reduced circulating lymphocyte count is below thenormal range and the subject is lymphopenic. Preferably, the subjectderives a therapeutic or prophylactic benefit from the reducedcirculating lymphocyte count. Such subjects include those suffering froma hematologic disease, such as a hematologic cancer, those sufferingfrom an autoimmune disorder, and those requiring modulation of an immuneresponse such as patients suffering from diabetes or organ transplantrecipients. In a human subject, the circulating lymphocyte count, forexample, B-lymphocytes, T-lymphocytes or both, can drop from a normalrange to a lymphopenic range. In certain diseases the circulatinglymphocyte count is abnormally high. In such diseases, the circulatinglymphocyte count can be reduced to the normal range or to a lymphopenicstate.

In one embodiment, the present invention includes the use of one or morecompounds of the invention in the manufacture of a medicament thatprevents further aberrant proliferation, differentiation, or survival ofcells. For example, compounds of the invention may be useful inpreventing tumors from increasing in size or from reaching a metastaticstate. The subject compounds may be administered to halt the progressionor advancement of cancer or to induce tumor apoptosis or to inhibittumor angiogenesis. In addition, the instant invention includes use ofthe subject compounds to prevent a recurrence of cancer.

This invention further embraces the treatment or prevention of cellproliferative disorders such as hyperplasias, dysplasias andpre-cancerous lesions. Dysplasia is the earliest form of pre-cancerouslesion recognizable in a biopsy by a pathologist. The subject compoundsmay be administered for the purpose of preventing said hyperplasias,dysplasias or pre-cancerous lesions from continuing to expand or frombecoming cancerous. Examples of pre-cancerous lesions may occur in skin,esophageal tissue, breast and cervical intra-epithelial tissue.

“Combination therapy” includes the administration of the subjectcompounds in further combination with other biologically activeingredients (such as, but not limited to, a second and differentantineoplastic agent) and non-drug therapies (such as, but not limitedto, surgery or radiation treatment). For instance, the compounds of theinvention can be used in combination with other pharmaceutically activecompounds, preferably compounds that are able to enhance the effect ofthe compounds of the invention. The compounds of the invention can beadministered simultaneously (as a single preparation or separatepreparation) or sequentially to the other drug therapy. In general, acombination therapy envisions administration of two or more drugs duringa single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g., FGF-R1,GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g., CEK5, CEK8, EBK, ECK,EEK, EHK-1, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); Axl (e.g.,Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g., PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g., p43^(abl), ARG); BTK (e.g., ITK/EMT,TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate non-kinase biological targets or processes. Such targetsinclude histone deacetylases (HDAC), DNA methyltransferase (DNMT), heatshock proteins (e.g., HSP90), hedgehog pathway-related proteins (e.g.,sonic hedgehog, patched, smoothened) and proteosomes.

In a preferred embodiment, subject compounds may be combined withantineoplastic agents (e.g., small molecules, monoclonal antibodies,antisense RNA, and fusion proteins) that inhibit one or more biologicaltargets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent,Sprycel, Nexavar, Sorafinib, CNF2024, RG108, BMS387032, Affinitak,Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD184322,Obatodax, ABT737, GDC-0449, IPI-926, BMS833923, LDE225, PF-04449913 andAEE788. Such combinations may enhance therapeutic efficacy over efficacyachieved by any of the agents alone and may prevent or delay theappearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives (Mechlorethamine,cyclophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines(thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazinesand Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide),Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide andmetal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloidssuch as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxeland Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine andVinorelbine), and Camptothecan analogs (Irinotecan and Topotecan);anti-tumor antibiotics such as Chromomycins (Dactinomycin andPlicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin,Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibioticssuch as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such asfolic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed,Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine,Cytarabine, Capecitabine, and Gemcitabine), purine antagonists(6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors(Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine,Nelarabine and Pentostatin); topoisomerase inhibitors such astopoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase IIinhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide);monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab,Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab,Bevacizumab); and miscellaneous anti-neoplastics such as ribonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor(Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubuleagents (Estramustine); retinoids (Bexarotene, Isotretinoin, Tretinoin(ATRA), and Lenalidomide.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemoprotective agent.Chemoprotective agents act to protect the body or minimize the sideeffects of chemotherapy. Examples of such agents include, but are notlimited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

It will be appreciated that compounds of the invention can be used incombination with an immunotherapeutic agent. One form of immunotherapyis the generation of an active systemic tumor-specific immune responseof host origin by administering a vaccine composition at a site distantfrom the tumor. Various types of vaccines have been proposed, includingisolated tumor-antigen vaccines and anti-idiotype vaccines. Anotherapproach is to use tumor cells from the subject to be treated, or aderivative of such cells (reviewed by Schirrmacher et al., (1995) J.Cancer Res. Clin. Oncol. 12 1:487). In U.S. Pat. No. 5,484,596, HannaJr., et al. claim a method for treating a resectable carcinoma toprevent recurrence or metastases, comprising surgically removing thetumor, dispersing the cells with collagenase, irradiating the cells, andvaccinating the patient with at least three consecutive doses of about10⁷ cells. It will be appreciated that the compounds of the inventionmay advantageously be used in conjunction with one or more adjunctivetherapeutic agents. Examples of suitable agents for adjunctive therapyinclude a 5HT₁ agonist, such as a triptan (e.g., sumatriptan ornaratriptan); an adenosine A1 agonist; an EP ligand; an NMDA modulator,such as a glycine antagonist; a sodium channel blocker (e.g.,lamotrigine); a substance P antagonist (e.g., an NK₁ antagonist); acannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; aleukotriene receptor antagonist; a DMARD (e.g., methotrexate);gabapentin and related compounds; a tricyclic antidepressant (e.g.,amitryptilline); a neuron stabilising antiepileptic drug; amono-aminergic uptake inhibitor (e.g., venlafaxine); a matrixmetalloproteinase inhibitor; a nitric oxide synthase (NOS) inhibitor,such as an iNOS or an nNOS inhibitor; an inhibitor of the release, oraction, of tumour necrosis factor .alpha.; an antibody therapy, such asa monoclonal antibody therapy; an antiviral agent, such as a nucleosideinhibitor (e.g., lamivudine) or an immune system modulator (e.g.,interferon); an opioid analgesic; a local anaesthetic; a stimulant,including caffeine; an H₂-antagonist (e.g., ranitidine); a proton pumpinhibitor (e.g., omeprazole); an antacid (e.g. aluminium or magnesiumhydroxide); an antiflatulent (e.g., simethicone); a decongestant (e.g.,phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline,epinephrine, naphazoline, xylometazoline, propylhexedrine, orlevo-desoxyephedrine); an antitussive (e.g., codeine, hydrocodone,carmiphen, carbetapentane, or dextramethorphan); a diuretic; or asedating or non-sedating antihistamine.

The compounds may also be used in the treatment of a disorder involving,relating to or, associated with dysregulation of histone deacetylase(HDAC). There are a number of disorders that have been implicated by orknown to be mediated at least in part by HDAC activity, where HDACactivity is known to play a role in triggering disease onset, or whosesymptoms are known or have been shown to be alleviated by HDACinhibitors. Disorders of this type that would be expected to be amenableto treatment with the compounds of the invention include the followingbut not limited to: Anti-proliferative disorders (e.g., cancers);Neurodegenerative diseases including Huntington's Disease, Polyglutaminedisease, Parkinson's Disease, Alzheimer's Disease, Seizures,Striatonigral degeneration, Progressive supranuclear palsy, Torsiondystonia, Spasmodic torticollis and dyskinesis, Familial tremor, Gillesde la Tourette syndrome, Diffuse Lewy body disease, Progressivesupranuclear palsy, Pick's disease, intracerebral hemorrhage, Primarylateral sclerosis, Spinal muscular atrophy, Amyotrophic lateralsclerosis, Hypertrophic interstitial polyneuropathy, Retinitispigmentosa, Hereditary optic atrophy, Hereditary spastic paraplegia,Progressive ataxia and Shy-Drager syndrome; Metabolic diseases includingType 2 diabetes; Degenerative Diseases of the Eye including Glaucoma,Age-related macular degeneration, Rubeotic glaucoma; Inflammatorydiseases and/or Immune system disorders including Rheumatoid Arthritis(RA), Osteoarthritis, Juvenile chronic arthritis, Graft versus Hostdisease, Psoriasis, Asthma, Spondyloarthropathy, Crohn's Disease,inflammatory bowel disease Colitis Ulcerosa, Alcoholic hepatitis,Diabetes, Sjoegrens's syndrome, Multiple Sclerosis, Ankylosingspondylitis, Membranous glomerulopathy, Discogenic pain, Systemic LupusErythematosus; Disease involving angiogenesis including cancer,psoriasis, rheumatoid arthritis; Psychological disorders includingbipolar disease, schizophrenia, mania, depression and dementia;Cardiovascular Diseases including the prevention and treatment ofischemia-related or reperfusion-related vascular and myocardial tissuedamage, heart failure, restenosis and arteriosclerosis; Fibroticdiseases including liver fibrosis, cystic fibrosis and angiofibroma;Infectious diseases including Fungal infections, such as candidiasis orCandida Albicans, Bacterial infections, Viral infections, such as HerpesSimplex, poliovirus, rhinovirus and coxsackievirus, Protozoalinfections, such as Malaria, Leishmania infection, Trypanosoma bruceiinfection, Toxoplasmosis and coccidlosis and Haematopoietic disordersincluding thalassemia, anemia and sickle cell anemia.

The compounds of the invention can also be used in the treatment of adisorder involving, relating to or, associated with dysregulation of PI3kinase. PI3 kinase activity has been implicated in or shown to beinvolved in a variety of disorders. In certain cases, PI3 kinaseactivity is involved in triggering disease onset, while in others,symptoms are known or have been shown to be alleviated by inhibitors ofPI3 kinase activity. Disorders of this type that would be expected to beamenable to treatment with the compounds of the invention include butare not limited to cancers, including leukemia, skin cancer, bladdercancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer,lung cancer, colon cancer, pancreatic cancer, renal cancer, gastriccancer and brain cancer; restenosis, atherosclerosis, bone disorders,arthritis, diabetic retinopathy, psoriasis, benign prostatichypertrophy, atherosclerosis, inflammation, angiogenesis, immunologicaldisorders, pancreatitis and kidney disease.

In one embodiment, compounds of the invention can be used to induce orinhibit apoptosis, a physiological cell death process critical fornormal development and homeostasis. Alterations of apoptotic pathwayscontribute to the pathogenesis of a variety of human diseases. Compoundsof the invention, as modulators of apoptosis, will be useful in thetreatment of a variety of human diseases with aberrations in apoptosisincluding cancer (particularly, but not limited to, follicularlymphomas, carcinomas with p53 mutations, hormone dependent tumors ofthe breast, prostate and ovary, and precancerous lesions such asfamilial adenomatous polyposis), viral infections (including, but notlimited to, herpes virus, poxvirus, Epstein-Barr virus, Sindbis virusand adenovirus), autoimmune diseases (including, but not limited to,systemic lupus, erythematosus, immune mediated glomerulonephritis,rheumatoid arthritis, psoriasis, inflammatory bowel diseases, andautoimmune diabetes mellitus), neurodegenerative disorders (including,but not limited to, Alzheimer's disease, AIDS-related dementia,Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, spinal muscular atrophy and cerebellar degeneration), AIDS,myelodysplastic syndromes, aplastic anemia, ischemic injury associatedmyocardial infarctions, stroke and reperfusion injury, arrhythmia,atherosclerosis, toxin-induced or alcohol induced liver diseases,hematological diseases (including, but not limited to, chronic anemiaand aplastic anemia), degenerative diseases of the musculoskeletalsystem (including, but not limited to, osteoporosis and arthritis),aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis,kidney diseases, and cancer pain.

In one aspect, the invention provides the use of compounds of theinvention for the treatment and/or prevention of immune response orimmune-mediated responses and diseases, such as the prevention ortreatment of rejection following transplantation of synthetic or organicgrafting materials, cells, organs or tissue to replace all or part ofthe function of tissues, such as heart, kidney, liver, bone marrow,skin, cornea, vessels, lung, pancreas, intestine, limb, muscle, nervetissue, duodenum, small-bowel, pancreatic-islet-cell, includingxeno-transplants, etc; to treat or prevent graft-versus-host disease,autoimmune diseases, such as rheumatoid arthritis, systemic lupuserythematosus, thyroiditis, Hashimoto's thyroiditis, multiple sclerosis,myasthenia gravis, type I diabetes uveitis, juvenile-onset orrecent-onset diabetes mellitus, uveitis, Graves disease, psoriasis,atopic dermatitis, Crohn's disease, ulcerative colitis, vasculitis,auto-antibody mediated diseases, aplastic anemia, Evan's syndrome,autoimmune hemolytic anemia, and the like; and further to treatinfectious diseases causing aberrant immune response and/or activation,such as traumatic or pathogen induced immune disregulation, includingfor example, that which are caused by hepatitis B and C infections, HIV,staphylococcus aureus infection, viral encephalitis, sepsis, parasiticdiseases wherein damage is induced by an inflammatory response (e.g.,leprosy); and to prevent or treat circulatory diseases, such asarteriosclerosis, atherosclerosis, vasculitis, polyarteritis nodosa andmyocarditis. In addition, the present invention may be used toprevent/suppress an immune response associated with a gene therapytreatment, such as the introduction of foreign genes into autologouscells and expression of the encoded product. Thus in one embodiment, theinvention relates to a method of treating an immune response disease ordisorder or an immune-mediated response or disorder in a subject in needof treatment comprising administering to said subject a therapeuticallyeffective amount of a compound of the invention.

In one aspect, the invention provides the use of compounds of theinvention in the treatment of a variety of neurodegenerative diseases, anon-exhaustive list of which includes: I. Disorders characterized byprogressive dementia in the absence of other prominent neurologic signs,such as Alzheimer's disease; Senile dementia of the Alzheimer type; andPick's disease (lobar atrophy); II. Syndromes combining progressivedementia with other prominent neurologic abnormalities such as: A)syndromes appearing mainly in adults (e.g., Huntington's disease,Multiple system atrophy combining dementia with ataxia and/ormanifestations of Parkinson's disease, Progressive supranuclear palsy(Steel-Richardson-Olszewski), diffuse Lewy body disease, andcorticodentatonigral degeneration; and B) syndromes appearing mainly inchildren or young adults (e.g., Hallervorden-Spatz disease andprogressive familial myoclonic epilepsy); III. Syndromes of graduallydeveloping abnormalities of posture and movement such as paralysisagitans (Parkinson's disease), striatonigral degeneration, progressivesupranuclear palsy, torsion dystonia (torsion spasm; dystonia musculorumdeformans), spasmodic torticollis and other dyskinesis, familial tremor,and Gilles de la Tourette syndrome; IV. Syndromes of progressive ataxiasuch as cerebellar degenerations (e.g., cerebellar cortical degenerationand olivopontocerebellar atrophy (OPCA)); and spinocerebellardegeneration (Friedreich's atazia and related disorders); V. Syndrome ofcentral autonomic nervous system failure (Shy-Drager syndrome); VI.Syndromes of muscular weakness and wasting without sensory changes(motorneuron disease such as amyotrophic lateral sclerosis, spinalmuscular atrophy (e.g., infantile spinal muscular atrophy(Werdnig-Hoffman), juvenile spinal muscular atrophy(Wohlfart-Kugelberg-Welander) and other forms of familial spinalmuscular atrophy), primary lateral sclerosis, and hereditary spasticparaplegia; VII. Syndromes combining muscular weakness and wasting withsensory changes (progressive neural muscular atrophy; chronic familialpolyneuropathies) such as peroneal muscular atrophy(Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy(Dejerine-Sottas), and miscellaneous forms of chronic progressiveneuropathy; VIII. Syndromes of progressive visual loss such aspigmentary degeneration of the retina (retinitis pigmentosa), andhereditary optic atrophy (Leber's disease). Furthermore, compounds ofthe invention can be implicated in chromatin remodeling.

The invention encompasses pharmaceutical compositions comprisingpharmaceutically acceptable salts of the compounds of the invention asdescribed above. The invention also encompasses solvates of thecompounds of the invention and pharmaceutical compositions comprisingsuch solvates, such as hydrates, methanolates or ethanolates. The term“solvate” refers to a solid, preferably crystalline, form of a compoundwhich includes the presence of solvent molecules within the crystallattice. A solvate of a compound comprising a given solvent is typicallyprepared by crystallization of the compound from that solvent. Solvatescan include a variety of solvents, including water, methanol andethanol. The term “hydrate” refers to a solvate in which the solvent iswater, and includes, but is not limited to, hemihydrate, monohydrate,dihydrate, trihydrate and the like. The invention further encompassespharmaceutical compositions comprising any solid or liquid physical formof the compound of the invention, including crystalline and crystallinesolvate forms. For example, the compounds can be in a crystalline form,in an amorphous form, and have any particle size. The particles may bemicronized, or may be agglomerated, particulate granules, powders, oils,oily suspensions or any other form of solid or liquid physical form.

The compounds of the invention, and derivatives, fragments, analogs,homologs, pharmaceutically acceptable salts or solvates thereof can beincorporated into pharmaceutical compositions suitable foradministration, together with a pharmaceutically acceptable carrier orexcipient. Such compositions typically comprise a therapeuticallyeffective amount of any of the compounds above, and a pharmaceuticallyacceptable carrier. Preferably, the effective amount when treatingcancer is an amount effective to selectively induce terminaldifferentiation of suitable neoplastic cells and less than an amountwhich causes toxicity in a patient.

Compounds of the invention may be administered by any suitable means,including, without limitation, parenteral, intravenous, intramuscular,subcutaneous, implantation, oral, sublingual, buccal, nasal, pulmonary,transdermal, topical, vaginal, rectal, and transmucosal administrationsor the like. Topical administration can also involve the use oftransdermal administration such as transdermal patches or iontophoresisdevices. Pharmaceutical preparations include a solid, semisolid orliquid preparation (tablet, pellet, troche, capsule, suppository, cream,ointment, aerosol, powder, liquid, emulsion, suspension, syrup,injection, etc.) containing a compound of the invention as an activeingredient, which is suitable for selected mode of administration. Inone embodiment, the pharmaceutical compositions are administered orally,and are thus formulated in a form suitable for oral administration,i.e., as a solid or a liquid preparation. Suitable solid oralformulations include tablets, capsules, pills, granules, pellets,sachets and effervescent, powders, and the like. Suitable liquid oralformulations include solutions, suspensions, dispersions, emulsions,oils and the like. In one embodiment of the present invention, thecomposition is formulated in a capsule. In accordance with thisembodiment, the compositions of the present invention comprise inaddition to the active compound and the inert carrier or diluent, a hardgelatin capsule.

Any inert excipient that is commonly used as a carrier or diluent may beused in the formulations of the present invention, such as for example,a gum, a starch, a sugar, a cellulosic material, an acrylate, ormixtures thereof. A preferred diluent is microcrystalline cellulose. Thecompositions may further comprise a disintegrating agent (e.g.,croscarmellose sodium) and a lubricant (e.g., magnesium stearate), andmay additionally comprise one or more additives selected from a binder,a buffer, a protease inhibitor, a surfactant, a solubilizing agent, aplasticizer, an emulsifier, a stabilizing agent, a viscosity increasingagent, a sweetener, a film forming agent, or any combination thereof.Furthermore, the compositions of the present invention may be in theform of controlled release or immediate release formulations.

For liquid formulations, pharmaceutically acceptable carriers may beaqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil. Solutions or suspensions can also include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

In addition, the compositions may further comprise binders (e.g.,acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g., cornstarch, potato starch, alginic acid,silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodiumstarch glycolate, Primogel), buffers (e.g., tris-HCI., acetate,phosphate) of various pH and ionic strength, additives such as albuminor gelatin to prevent absorption to surfaces, detergents (e.g., Tween20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors,surfactants (e.g., sodium lauryl sulfate), permeation enhancers,solubilizing agents (e.g., glycerol, polyethylene glycerol, polyethyleneglycol), a glidant (e.g., colloidal silicon dioxide), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole),stabilizers (e.g., hydroxypropyl cellulose, hydroxypropylmethylcellulose), viscosity increasing agents (e.g., carbomer, colloidalsilicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose,aspartame, citric acid), flavoring agents (e.g., peppermint, methylsalicylate, or orange flavoring), preservatives (e.g., Thimerosal,benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesiumstearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g.,colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate,triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose,sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g., ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

Formulations of the invention intended for oral administration caninclude one or more permeation enhancers, including long chain fattyacids or salts thereof, such as decanoic acid and sodium decanoate.

In one preferred embodiment, the compound can be formulated in anaqueous solution for intravenous injection. In one embodiment,solubilizing agents can be suitably employed. A particularly preferredsolubilizing agent includes cyclodextrins and modified cyclodextrins,such as sulfonic acid substituted β-cyclodextrin derivative or saltthereof, including sulfobutyl derivatized-β-cyclodextrin, such assulfobutylether-7-β-cyclodextrin which is sold by CyDex, Inc. under thetradename CAPTISOL®.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Daily administration may be repeated continuously for a period ofseveral days to several years. Oral treatment may continue for betweenone week and the life of the patient. Preferably the administration maytake place for five consecutive days after which time the patient can beevaluated to determine if further administration is required. Theadministration can be continuous or intermittent, e.g., treatment for anumber of consecutive days followed by a rest period. The compounds ofthe present invention may be administered intravenously on the first dayof treatment, with oral administration on the second day and allconsecutive days thereafter.

The preparation of pharmaceutical compositions that contain an activecomponent is well understood in the art, for example, by mixing,granulating, or tablet-forming processes. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active agents are mixed with additives customary forthis purpose, such as vehicles, stabilizers, or inert diluents, andconverted by customary methods into suitable forms for administration,such as tablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions and the like as detailed above.

The amount of the compound administered to the patient is less than anamount that would cause toxicity in the patient. In certain embodiments,the amount of the compound that is administered to the patient is lessthan the amount that causes a concentration of the compound in thepatient's plasma to equal or exceed the toxic level of the compound.Preferably, the concentration of the compound in the patient's plasma ismaintained at about 10 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 25 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 50 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 100 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 500 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 1000 nM. In oneembodiment, the concentration of the compound in the patient's plasma ismaintained at about 2500 nM. In one embodiment, the concentration of thecompound in the patient's plasma is maintained at about 5000 nM. Theoptimal amount of the compound that should be administered to thepatient in the practice of the present invention will depend on theparticular compound used and the type of cancer being treated.

Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “acyl” refers to hydrogen, alkyl, partially saturated or fullysaturated cycloalkyl, partially saturated or fully saturatedheterocycle, aryl, and heteroaryl substituted carbonyl groups. Forexample, acyl includes groups such as (C₁-C₆)alkanoyl (e.g., formyl,acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.),(C₃-C₆)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl,tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl(e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl,furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl,cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl groupmay be any one of the groups described in the respective definitions.When indicated as being “optionally substituted”, the acyl group may beunsubstituted or optionally substituted with one or more substituents(typically, one to three substituents) independently selected from thegroup of substituents listed below in the definition for “substituted”or the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion ofthe acyl group may be substituted as described above in the preferredand more preferred list of substituents, respectively.

The term “alkyl” embraces linear or branched radicals having one toabout twenty carbon atoms or, preferably, one to about twelve carbonatoms. More preferred alkyl radicals are “lower alkyl” radicals havingone to about ten carbon atoms. Most preferred are lower alkyl radicalshaving one to about eight carbon atoms. Examples of such radicalsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twenty carbon atoms or,preferably, two to about twelve carbon atoms. More preferred alkenylradicals are “lower alkenyl” radicals having two to about ten carbonatoms and more preferably about two to about eight carbon atoms.Examples of alkenyl radicals include ethenyl, allyl, propenyl, butenyland 4-methylbutenyl. The terms “alkenyl”, and “lower alkenyl”, embraceradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations.

The term “alkynyl” embraces linear or branched radicals having at leastone carbon-carbon triple bond of two to about twenty carbon atoms or,preferably, two to about twelve carbon atoms. More preferred alkynylradicals are “lower alkynyl” radicals having two to about ten carbonatoms and more preferably about two to about eight carbon atoms.Examples of alkynyl radicals include propargyl, 1-propynyl, 2-propynyl,1-butyne, 2-butynyl and 1-pentynyl.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one, two or three rings wherein such rings may beattached together in a pendent manner or may be fused. The term “aryl”embraces aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl,indane and biphenyl.

The terms “heterocyclyl”, “heterocycle”, “heterocyclic” or “heterocyclo”embrace saturated, partially unsaturated and unsaturatedheteroatom-containing ring-shaped radicals, which can also be called“heterocyclyl”, “heterocycloalkenyl” and “heteroaryl” correspondingly,where the heteroatoms may be selected from nitrogen, sulfur and oxygen.Examples of saturated heterocyclyl radicals include saturated 3 to6-membered heteromonocyclic group containing 1 to 4 nitrogen atoms (e.g.pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.); saturated3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atomsand 1 to 3 nitrogen atoms (e.g. morpholinyl, etc.); saturated 3 to6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1to 3 nitrogen atoms (e.g., thiazolidinyl, etc.). Examples of partiallyunsaturated heterocyclyl radicals include dihydrothiophene,dihydropyran, dihydrofuran and dihydrothiazole. Heterocyclyl radicalsmay include a pentavalent nitrogen, such as in tetrazolium andpyridinium radicals. The term “heterocycle” also embraces radicals whereheterocyclyl radicals are fused with aryl or cycloalkyl radicals.Examples of such fused bicyclic radicals include benzofuran,benzothiophene, and the like.

The term “heteroaryl” embraces unsaturated heterocyclyl radicals.Examples of heteroaryl radicals include unsaturated 3 to 6-membered,preferably 5 or 6-membered, heteromonocyclic group containing 1 to 4nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl,pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g.,4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.)tetrazolyl (e.g. 1H-tetrazolyl, 2H-tetrazolyl, etc.), etc.; unsaturatedcondensed heterocyclyl group containing 1 to 5 nitrogen atoms, forexample, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl,isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g.,tetrazolo[1,5-b]pyridazinyl, etc.), etc.; unsaturated 3 to 6-membered,preferably 5- or 6-membered, heteromonocyclic group containing an oxygenatom, for example, pyranyl, furyl, etc.; unsaturated 3 to 6-memberedheteromonocyclic group containing a sulfur atom, for example, thienyl,etc.; unsaturated 3 to 6-membered, preferably 5- or 6-membered,heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl (e.g.,1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.) etc.;unsaturated condensed heterocyclyl group containing 1 to 2 oxygen atomsand 1 to 3 nitrogen atoms (e.g. benzoxazolyl, benzoxadiazolyl, etc.);unsaturated 3 to 6-membered, preferably 5- or 6-membered,heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, for example, thiazolyl, thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.) etc.;unsaturated condensed heterocyclyl group containing 1 to 2 sulfur atomsand 1 to 3 nitrogen atoms (e.g., benzothiazolyl, benzothiadiazolyl,etc.) and the like.

The term “heterocycloalkyl” embraces heterocyclo-substituted alkylradicals. More preferred heterocycloalkyl radicals are “lowerheterocycloalkyl” radicals having one to six carbon atoms in theheterocyclo radicals.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl,arylthioalkyl, alkyl sulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl,alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino,trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,heteroaryl, heterocyclic, and aliphatic. It is understood that thesubstituent may be further substituted.

For simplicity, chemical moieties are defined and referred to throughoutcan be univalent chemical moieties (e.g., alkyl, aryl, etc.) ormultivalent moieties under the appropriate structural circumstancesclear to those skilled in the art. For example, an “alkyl” moiety can bereferred to a monovalent radical (e.g., CH₃—CH₂—), or in otherinstances, a bivalent linking moiety can be “alkyl,” in which case thoseskilled in the art will understand the alkyl to be a divalent radical(e.g., —CH₂—CH₂—), which is equivalent to the term “alkylene.”Similarly, in circumstances in which divalent moieties are required andare stated as being “alkoxy”, “alkylamino”, “aryloxy”, “alkylthio”,“aryl”, “heteroaryl”, “heterocyclic”, “alkyl” “alkenyl”, “alkynyl”,“aliphatic”, or “cycloalkyl”, those skilled in the art will understandthat the terms alkoxy“, “alkylamino”, “aryloxy”, “alkylthio”, “aryl”,“heteroaryl”, “heterocyclic”, “alkyl”, “alkenyl”, “alkynyl”,“aliphatic”, or “cycloalkyl” refer to the corresponding divalent moiety.

The terms “halogen” or “halo” as used herein, refers to an atom selectedfrom fluorine, chlorine, bromine and iodine.

As used herein, the term “aberrant proliferation” refers to abnormalcell growth.

The phrase “adjunctive therapy” encompasses treatment of a subject withagents that reduce or avoid side effects associated with the combinationtherapy of the present invention, including, but not limited to, thoseagents, for example, that reduce the toxic effect of anticancer drugs,e.g., bone resorption inhibitors, cardioprotective agents; prevent orreduce the incidence of nausea and vomiting associated withchemotherapy, radiotherapy or operation; or reduce the incidence ofinfection associated with the administration of myelosuppressiveanticancer drugs.

The term “angiogenesis,” as used herein, refers to the formation ofblood vessels. Specifically, angiogenesis is a multi-step process inwhich endothelial cells focally degrade and invade through their ownbasement membrane, migrate through interstitial stroma toward anangiogenic stimulus, proliferate proximal to the migrating tip, organizeinto blood vessels, and reattach to newly synthesized basement membrane(see Folkman et al., Adv. Cancer Res., Vol. 43, pp. 175-203 (1985)).Anti-angiogenic agents interfere with this process. Examples of agentsthat interfere with several of these steps include thrombospondin-1,angiostatin, endostatin, interferon alpha and compounds such as matrixmetalloproteinase (MMP) inhibitors that block the actions of enzymesthat clear and create paths for newly forming blood vessels to follow;compounds, such as .alpha.v.beta.3 inhibitors, that interfere withmolecules that blood vessel cells use to bridge between a parent bloodvessel and a tumor; agents, such as specific COX-2 inhibitors, thatprevent the growth of cells that form new blood vessels; andprotein-based compounds that simultaneously interfere with several ofthese targets.

The term “apoptosis” as used herein refers to programmed cell death assignaled by the nuclei in normally functioning human and animal cellswhen age or state of cell health and condition dictates. An “apoptosisinducing agent” triggers the process of programmed cell death.

The term “cancer” as used herein denotes a class of diseases ordisorders characterized by uncontrolled division of cells and theability of these cells to invade other tissues, either by direct growthinto adjacent tissue through invasion or by implantation into distantsites by metastasis.

The terms “compound” and “compound of the invention”, as used herein,refer to compounds of Formula I and pharmaceutically acceptable saltsthereof. The compounds of the invention can be obtained in differentforms, including crystalline and amorphous forms. The compounds can alsooccur as solvates, for example, hydrates, or solvates of an organicsolvent, preferably a pharmaceutically acceptable solvent. The compoundscan also occur in multiple crystalline, or polymorphic, forms. Thecompounds of the invention further include pharmaceutically acceptableprodrugs and esters of the compounds of Formula I.

The term “device” refers to any appliance, usually mechanical orelectrical, designed to perform a particular function.

As used herein, the term “dysplasia” refers to abnormal cell growth, andtypically refers to the earliest form of pre-cancerous lesionrecognizable in a biopsy by a pathologist.

As used herein, the term “effective amount of the subject compounds,”with respect to the subject method of treatment, refers to an amount ofthe subject compound which, when delivered as part of desired doseregimen, brings about, e.g., a change in the rate of cell proliferationand/or state of differentiation and/or rate of survival of a cell toclinically acceptable standards. This amount may further relieve to someextent one or more of the symptoms of a neoplasia disorder, including,but is not limited to: 1) reduction in the number of cancer cells; 2)reduction in tumor size; 3) inhibition (i.e., slowing to some extent,preferably stopping) of cancer cell infiltration into peripheral organs;4) inhibition (i.e., slowing to some extent, preferably stopping) oftumor metastasis; 5) inhibition, to some extent, of tumor growth; 6)relieving or reducing to some extent one or more of the symptomsassociated with the disorder; and/or 7) relieving or reducing the sideeffects associated with the administration of anticancer agents.

The term “hyperplasia,” as used herein, refers to excessive celldivision or growth.

The phrase an “immunotherapeutic agent” refers to agents used totransfer the immunity of an immune donor, e.g., another person or ananimal, to a host by inoculation.

The term embraces the use of serum or gamma globulin containingperformed antibodies produced by another individual or an animal;nonspecific systemic stimulation; adjuvants; active specificimmunotherapy; and adoptive immunotherapy. Adoptive immunotherapy refersto the treatment of a disease by therapy or agents that include hostinoculation of sensitized lymphocytes, transfer factor, immune RNA, orantibodies in serum or gamma globulin.

The term “inhibition,” in the context of neoplasia, tumor growth ortumor cell growth, may be assessed by delayed appearance of primary orsecondary tumors, slowed development of primary or secondary tumors,decreased occurrence of primary or secondary tumors, slowed or decreasedseverity of secondary effects of disease, arrested tumor growth andregression of tumors, among others. In the extreme, complete inhibition,is referred to herein as prevention or chemoprevention.

The term “metastasis,” as used herein, refers to the migration of cancercells from the original tumor site through the blood and lymph vesselsto produce cancers in other tissues. Metastasis also is the term usedfor a secondary cancer growing at a distant site.

The term “neoplasm,” as used herein, refers to an abnormal mass oftissue that results from excessive cell division. Neoplasms may bebenign (not cancerous), or malignant (cancerous) and may also be calleda tumor. The term “neoplasia” is the pathological process that resultsin tumor formation.

As used herein, the term “pre-cancerous” refers to a condition that isnot malignant, but is likely to become malignant if left untreated.

The term “proliferation” refers to cells undergoing mitosis.

The phrase “PI3 kinase related disease or disorder” refers to a diseaseor disorder characterized by inappropriate phosphoinositide-3-kinaseactivity or over-activity of the phosphoinositide-3-kinase.Inappropriate activity refers to either: (i) PI3 kinase expression incells which normally do not express PI3 kinase; (ii) increased PI3kinase expression leading to unwanted cell proliferation,differentiation and/or growth; or, (iii) decreased PI3 kinase expressionleading to unwanted reductions in cell proliferation, differentiationand/or growth. Over-activity of PI3 kinase refers to eitheramplification of the gene encoding a particular PI3 kinase or productionof a level of PI3 kinase activity which can correlate with a cellproliferation, differentiation and/or growth disorder (that is, as thelevel of the PI3 kinase increases, the severity of one or more of thesymptoms of the cellular disorder increases).

The phrase a “radio therapeutic agent” refers to the use ofelectromagnetic or particulate radiation in the treatment of neoplasia.

The term “recurrence” as used herein refers to the return of cancerafter a period of remission. This may be due to incomplete removal ofcells from the initial cancer and may occur locally (the same site ofinitial cancer), regionally (in vicinity of initial cancer, possibly inthe lymph nodes or tissue), and/or distally as a result of metastasis.

The term “treatment” refers to any process, action, application,therapy, or the like, wherein a mammal, including a human being, issubject to medical aid with the object of improving the mammal'scondition, directly or indirectly.

The term “vaccine” includes agents that induce the patient's immunesystem to mount an immune response against the tumor by attacking cellsthat express tumor associated antigens (Teas).

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid or inorganic acid. Examples of pharmaceuticallyacceptable nontoxic acid addition salts include, but are not limited to,salts of an amino group formed with inorganic acids such as hydrochloricacid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloricacid or with organic acids such as acetic acid, maleic acid, tartaricacid, citric acid, succinic acid lactobionic acid or malonic acid or byusing other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include, but are not limited to,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl sulfonate having from 1 to 6 carbon atoms, sulfonate andaryl sulfonate. Certain salts such as the sodium, potassium and cholinebase salts as well as acidic salts such as sulfate and methanesulfonatesalts have been found to improve the solubility of compounds of FormulaI in pharmaceutically acceptable aqueous media. In one embodiment, thepharmaceutically acceptable salt of Compound 1 is the choline salt.Preferred salts of Compound 1 include the sodium salt and the potassiumsalt. Other preferred salts include the sulfate and methanesulfonatesalts.

As used herein, the term “pharmaceutically acceptable ester” refers toesters which hydrolyze in vivo and include those that break down readilyin the human body to leave the parent compound or a salt thereof.Suitable ester groups include, for example, those derived frompharmaceutically acceptable aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.Examples of particular esters include, but are not limited to, formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals with undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of thepresent invention. “Prodrug”, as used herein means a compound which isconvertible in vivo by metabolic means (e.g. by hydrolysis) to acompound of the invention. Various forms of prodrugs are known in theart, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs,Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, Vol. 4,Academic Press (1985); Krogsgaard-Larsen, et al., (ed.), “Design andApplication of Prodrugs, Textbook of Drug Design and Development,Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug DeliverReviews, 8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel DrugDelivery Systems, American Chemical Society (1975); and Bernard Testa &Joachim Mayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry,Biochemistry And Enzymology, John Wiley and Sons, Ltd. (2002).

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration, such as sterilepyrogen-free water. Suitable carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, a standard referencetext in the field, which is incorporated herein by reference. Preferredexamples of such carriers or diluents include, but are not limited to,water, saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

As used herein, the term “pre-cancerous” refers to a condition that isnot malignant, but is likely to become malignant if left untreated.

The term “subject” as used herein refers to an animal. Preferably theanimal is a mammal. More preferably the mammal is a human. A subjectalso refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, fish, birds and the like.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and may include those which increasebiological penetration into a given biological system (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the inventionformulated together with one or more pharmaceutically acceptablecarriers or excipients.

Preferably, the pharmaceutically acceptable carrier or excipient is anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as lactose, glucose and sucrose; cyclodextrinssuch as alpha-(α), beta-(β) and gamma-(γ) cyclodextrins; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycolssuch as propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or: a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid; b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate; h) absorbents such as kaolinand bentonite clay; and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

For pulmonary delivery, a therapeutic composition of the invention isformulated and administered to the patient in solid or liquidparticulate form by direct administration (e.g., inhalation into therespiratory system). Solid or liquid particulate forms of the activecompound prepared for practicing the present invention include particlesof respirable size: that is, particles of a size sufficiently small topass through the mouth and larynx upon inhalation and into the bronchiand alveoli of the lungs. Delivery of aerosolized therapeutics,particularly aerosolized antibiotics, is known in the art (see, forexample U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat. No.5,508,269 to Smith et al., and WO 98/43650 by Montgomery, all of whichare incorporated herein by reference). A discussion of pulmonarydelivery of antibiotics is also found in U.S. Pat. No. 6,014,969,incorporated herein by reference.

By a “therapeutically effective amount” of a compound of the inventionis meant an amount of the compound which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). An effective amount of the compounddescribed above may range from about 0.1 mg/Kg to about 500 mg/Kg,preferably from about 1 to about 50 mg/Kg. Effective doses will alsovary depending on route of administration, as well as the possibility ofco-usage with other agents. It will be understood, however, that thetotal daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or contemporaneously with thespecific compound employed; and like factors well known in the medicalarts.

The total daily dose of the compounds of this invention administered toa human or other animal in single or in divided doses can be in amounts,for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1to 25 mg/kg body weight. Single dose compositions may contain suchamounts or submultiples thereof to make up the daily dose. In general,treatment regimens according to the present invention compriseadministration to a patient in need of such treatment from about 10 mgto about 1000 mg of the compound(s) of this invention per day in singleor multiple doses.

The compounds of the formulae described herein can, for example, beadministered by injection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.1 toabout 500 mg/kg of body weight, alternatively dosages between 1 mg and1000 mg/dose, every 4 to 120 hours, or according to the requirements ofthe particular drug. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositions ofthis invention will be administered from about 1 to about 6 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with pharmaceutically excipients or carriers toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. A typical preparation willcontain from about 5% to about 95% active compound (w/w). Alternatively,such preparations may contain from about 20% to about 80% activecompound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

EXAMPLES

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

The synthesis of Compound 1 and the methanesulfonate, sodium, potassiumand choline salts thereof is illustrated in the schemes below.

The intermediate 107-1 or 107-2 can be prepared by reacting 106 witheither R-2-1 or R-2-2, respectively. The synthetic schemes for thesynthesis of R-2-1 and R-2-2 are illustrated below:

Or by an alternative method:

Intermediate 108-1 and 108-2 can be prepared by the coupling reaction of107-1 or 107-2 with either R-3-1 or R-3-2, where R-3-1 and R-3-2 can beprepared according to the following scheme:

Example 1 Preparation ofN-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide(Compound 1) Step a: (Z)-Ethyl-2-(ethoxymethyl)-3-methoxyacrylate(Compound 202)

Sodium (40.9 g, 1.78 mol) was added to ethanol (750 mL) in portionscarefully and the solution was concentrated to give NaOEt powder afterall sodium metal disappeared. Under stirring, hexane (1.0 L) was addedand the mixture was cooled with ice-water bath. A mixture of 201 (130 g,0.89 mol) and ethyl formate (131 g, 1.78 mol) was added dropwise at 0-5°C. The reaction mixture was stirred at room temperature overnight.Dimethyl sulfate (224 g, 1.78 mol) was added dropwise with cooling ofice-water bath. The resulting mixture was heated at 50° C. for 2 h. Tothe mixture was added triethylammonium chloride (122 g) and sodiumhydroxide (20 g). The mixture was then stirred at room temperature for 4h and filtered. The filtrate was washed with water and dried overNa₂SO₄. It was concentrated to afford the titled compound (140 g, 37%)as a colorless oil which was used in the next step without furtherpurification.

Step b: Ethyl 2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (Compound203)

A mixture of compound 202 (140 g, 0.745 mol), urea (40.0 g, 0.697 mol)and concentrated hydrochloric acid (34 mL) in ethanol (500 mL) washeated at reflux overnight. After evaporating ˜50% of volume ofreaction, the resulting suspension was filtered, washed with smallamount of ethanol, and dried to give compound 203 (47 g, 37%) as a whitesolid. LCMS: 171 [M+1]⁺. ¹H NMR (400 MHz, CDCl₃): δ 1.19 (t, J=7.2 Hz ,3H), 3.92 (s, 2H), 4.08 (q, J=7.2 Hz, 2H), 7.0 (s, 1H), 7.08 (d, J=6.0Hz , 1H), 8.83 (d, br, J=4.8 Hz, 1H).

Step c: Ethyl 2-oxo-1,2-dihydropyrimidine-5-carboxylate (Compound 204)

To a solution of compound 203 (47 g, 280 mmol) in acetic acid (500 mL)was added bromine (49.0 g, 307 mmol). The mixture was heated at refluxfor 2 h, cooled to room temperature, further cooled at 0-5° C. andfiltered to give the title compound 204 as a yellow solid (38 g, 54%).LCMS: 169 [M+1]⁺. ¹H NMR (400 MHz, D₂O): δ 1.28 (t, J=7.2 Hz , 3H), 4.32(q, J=7.2 Hz , 2H), 9.00 (br, s, 2H).

Step d: Ethyl 2-chloropyrimidine-5-carboxylate (Compound R-2-1)

A mixture of compound 204 (38.0 g, 153 mmol) and phosphoryl trichloride(300 mL) and N, N-dimethylaniline (3 mL) was heated at reflux for 2 h,cooled to room temperature and concentrated. The residue was quenchedcarefully with ice-water, adjusted pH to 7-8 with sodium carbonate andextracted with EtOAc. The combined organics were washed with ice-waterand brine, dried over Na₂SO₄, evaporated, and purified by columnchromatography (eluted with EtOAc/Hexanes, 10%) to afford compound R-2-1(15 g, 52%) as a white solid. LCMS: 187 [M+1]⁺. ¹H NMR (400 MHz, CDCl₃):δ 1.36 (t, J=7.5 Hz , 3H), 4.39 (q, J=7.5 Hz, 2H), 9.08 (s, 2H).

Step e: Sodium (Z)-2-(dimethoxymethyl)-3-methoxy-3-oxoprop-1-en-1-olate(Compound 206)

A mixture of NaH (27 g, 60% in mineral oil, 0.675mo1) in anhydrous1,2-dimethoxyethane (300 mL) was heated to 40-50° C. and methyl3,3-dimethoxy propionate (205) (100 g, 0.675 mol) was added dropwise.The resulting mixture was stirred for 0.5 h and anhydrous methyl formate(81 g, 1.35 mol) was added dropwise at 40-50° C. The resulting mixturewas stirred at 40-50° C. (inner temperature) for 2 h before it wascooled to 0° C. The reaction mixture was allowed to warm to 25° C.slowly and stirred overnight. Et₂O (150 mL) was added and stirred for 30min. The resulting suspension was filtered. The solid was washed withEt₂O (100mL), collected and dried to afford the title compound 206 (82g, 61%) as an off-white solid. LCMS (m/z): 130.8 [M+1]⁺. ¹HNMR (400 MHz,CD₃OD): δ 3.36 (s, 6H), 3.60 (s, 3H), 5.34 (s, 1H), 8.92 (s, 1H).

Step f: 2-Amino-pyrimidine-5-carboxylic acid methyl ester (Compound 207)

To a mixture of guanidine hydrochloride (42.2 g, 0.44 mol) in DMF (300mL) was added compound 206 (80 g, 0.40 mol). The resulting mixture washeated at 100° C. for 1 h. The reaction mixture was filtered beforecooled. The filter cake was washed with 50 mL of DMF and the combinedfiltrate was concentrated to leave a residue which was suspended in coldEtOH and washed with cold EtOH (50 mL) to afford the compound 207 (38 g,61.5%) as a yellow solid. LCMS (m/z): 154.2 [M+1]⁺, 195.1[M+42]⁺. ¹H NMR(400 MHz, CD₃OD): δ 3.88 (s, 3H), 8.77 (s, 2H).

Step g: Methyl 2-chloropyrimidine-5-carboxylate (Compound R-2-2)

Compound 207 (7 g, 0.046 mol) was added to a mixture of concentratedhydrochloric acid (15.2 mL) and CH₂Cl₂(60 mL). After cooling, ZnCl₂(18.6 g, 0.138 mol) was added at 15-20° C. The mixture was stirred at15-20° C. for 0.5 h and cooled to 5-10° C. NaNO₂ (9.5 g, 0.138 mol) wasadded portion wise while keeping the internal temperature 5-10° C. Thereaction was continued for ˜2 h. The reaction mixture was poured intoice-water (50 mL). The organic layer was separated and the aqueous phasewas extracted with CH₂Cl₂ (30 mL*2). The combined organic extracts wereconcentrated to afford crude product (4.2 g). The crude compound wassuspended in hexane (20 mL), heated at 60° C. for 30 minutes andfiltered. The filtrate was concentrated to afford the title compoundR-2-2 (3.5 g, 44.4%) as an off-white solid. LCMS (m/z): 214.1[M+42]⁺. ¹HNMR (400 MHz, CDCl₃): δ 4.00 (s, 3H), 9.15 (s, 2H).

Step h: 5-Bromo-2-methoxypyridine (Compound 303)

A solution of 2-methoxy-pyridine (100 g, 0.92 mole), NBS (180 g, 1.0mole) in acetonitrile (1.0 L) was stirred at reflux for 21 h. TLC showedthat the reaction was complete. The reaction mixture was cooled to roomtemperature and concentrated. ˜900 ml solvent was collected. Theresulting suspension was filtered and washed with n-hexane (˜400mL). Thefiltrate was concentrated again to afford crude product. The crudeproduct was distilled at reduced pressure (30° C./˜0.3 mmHg) to affordthe title compound as a clear oil (146 g, 84%). LCMS (m/z): 190.0[M+1]⁺. ¹H NMR (400 MHz, CDCl₃): δ 3.90 (s, 3H), 6.65 (d, J=8.8 Hz, 1H),7.62 (dd, J=8.8 Hz, 2.4Hz, 1H), 8.19 (s, 1H).

Step i: 6-Methoxypyridin-3-ylboronic acid (R-3-1):

To a solution of compound 303 (20 g, 0.11 mole) in anhydrous THF (180ml) was added dropwise n-BuLi (59 mL, 2M in THF) at −78° C., theresulting mixture was stirred for 1 h. Triisopropyl borate (37 mL) wasadded at −78° C. and the reaction mixture was warmed to room temperatureand continued to stir overnight. TLC (hexanes/ethyl acetate=5:1) showedreaction complete. The mixture was adjusted pH to 3-4 with 4N HCl (90ml). The precipitate was collected by filtration to afford crudecompound R-3-1 (21 g, 128%). The crude compound R-3-1 (21 g) wasdissolved in water (200 ml) and the solution was adjusted pH to 8-9 withconcentrated ammonia solution, the precipitate was collected byfiltration to afford the pure title compound R-3-1 as a white solid.(11g, 67%). LCMS (m/z): 154.1 [M+1]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 3.86 (s,3H), 6.76 (d, J=8.4 Hz, 1H), 7.99 (dd, J=8.4 Hz, 2.0 Hz, 1H), 8.05 (br,2H), 8.52 (d, J=2.0 Hz, 1H).

Step j:2-methoxy-5-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine(Compound R-3-2)

A mixture of compound 303 (55 g, 0.29 mol),4,4,4′,4′,5,5,5′,5′-octamethyl -2,2′-bi(1,3,2-dioxaborolane) (90 g, 0.35mol), potassium acetate (57 g, 0.58 mol) andbis(triphenylphosphine)palladium(II) chloride (2.2 g, 3 mmol) inanhydrous dioxane (500 mL) was heated at 108° C. under N₂ atmosphereovernight. The reaction mixture was concentrated and purified by columnchromatography eluted with hexanes/ethyl acetate to afford titlecompound R-3-2 (58 g, 84%). ¹H NMR (400 MHz, DMSO-d₆): δ 1.30 (s, 12H),3.88 (s, 3H), 6.81 (d, J=8.0 Hz, 1H), 7.88 (dd, J=8.0 Hz, 2.0 Hz, 1H),8.41 (d, J=2.0 Hz, 1H).

Step k: Thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (Compound 102)

Urea method: A mixture of methyl 3-aminothiophene-2-carboxylate (101)(90.0 g, 573 mmol, 1.0 eq) and urea (277.6 g, 4.6 mol, 8.0 eq) washeated at 190° C. for 3-4 h and cooled to room temperature. To thereaction mixture was added aq. NaOH (10%, 800 mL). After stirring atambient temperature for 1 h, the solid was removed by filtration. Thefiltrate was acidified with HC1 to pH 3-4, the precipitated solid wascollected by filtration, washed with water and dried in vacuo to givethe desired product compound 102 as an off-white solid (87 g, 89%).m.p.:280-285° C. LCMS (m/z): 169.0 [M+1]⁺. ¹H NMR (400 MHz, DMSO- d₆): δ6.92 (d, J=5.2 Hz, 1H), 8.05 (d, J=5.2 Hz, 1H), 11.0-11.5 (br, 2H).

KOCN method: To a mixture of 3-aminothiophene-2-carboxylate (101) (100.0g, 636.9 mmol, 1.0 eq), acetic acid (705 mL) and water (600 mL) wasadded a solution of potassium cyanate (154.8 g, 1.91 mol, 3.0 eq) inwater (326 mL) slowly over a period of 1 h. The resulting mixture wasstirred at room temperature for 20 h, filtered and rinsed with water(500 mL). The cake was charged to a suitably sized reactor and 2 Maqueous sodium hydroxide solution (1.65 L) was added, the slurry wasstirred for 2 h and LCMS confirmed the formation of the desired product.The mixture was cooled to 10° C. and 3 M aqueous hydrochloric acid (1.29L) was added until the pH =5.0˜6.0. The slurry was filtered, rinsed withwater (700 mL), and dried in vacuum oven at 50° C. for 24 h to affordcompound 102 (100 g, 94%) as an off-white solid. LCMS (m/z): 169.1[M+1]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 6.92 (d, J=5.2 Hz, 1H), 8.04 (d,J=5.2 Hz, 1H), 11.14 (s, 1H), 11.51 (s, 1H).

Step 1: 2,4-Dichlorothieno[3,2-d]pyrimidine (Compound 103)

Phosphorous oxychloride (152 mL, 1.67 mol, 7.0 eq) was added slowly tocold solution of compound 102 (40 g, 238 mmol, 1.0 eq) andN,N-dimethylaniline (22.5 mL, 179 mmol, 0.75 eq) in acetonitrile (250mL) while maintaining the temperature below 20° C. The mixture was thenheated to 85° C. and stirred for 24 h. The reaction mixture was cooledto 15° C., and then poured slowly onto a mixture of ice and cold water(360 mL). The resulting slurry was filtered, rinsed with cold water (200mL). The cake was dried in vacuum oven at 40° C. for 24 h to affordcompound 103 (40.5 g, 83%) as an off- white solid. M.p.:245-250° C. LCMS(m/z): 205.0 [M+1]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 7.75 (d, J=5.2 Hz,1H), 8.71 (d, J=5.2 Hz, 1H).

Step m: 2-Chloro-4-morpholinothieno[3,2-d]pyrimidine (Compound 104)

To a mixture of compound 103 (34.2 g, 167 mmol, 1.0 eq) and methanol(500 mL) was added morpholine (31.2 mL, 367 mmol, 2.2 eq) slowly. Thereaction mixture was stirred at room temperature overnight. Theprecipitate was collected by filtration, washed with methanol and driedin vacuo to give the desired product compound 104 as a light-yellowsolid (39 g, 91%). M.p.: 250-255° C. LCMS (m/z): 256.0 [M+1]⁺. ¹H NMR(400 MHz, DMSO-d₆): δ 3.76 (t, J=5.2 Hz, 4H), 3.92 (t, J=5.2 Hz, 4H),7.42 (d, J=5.2 Hz, 1H), 8.32 (d, J=5.2 Hz, 1H).

Step n: 2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde(Compound 105)

To a suspension of compound 104 (20 g, 78.4 mmol, 1.0 eq) in THF(anhydrous, 320 mL) at −78° C. was added n-BuLi (in hexanes, 2.4 M, 40.8mL, 102 mmol, 1.3 eq) slowly under nitrogen. The resulting slurry wasallowed to warm up to −60° C. to turn into a clear brown solution. Thereaction mixture was then cooled to −78° C. again and DMF (anhydrous,9.1 mL, 118 mmol, 1.5 eq) was added slowly. The resulting solution wasstirred at −78° C. for 0.5 h, warmed up to 0° C. over 1 h and was pouredslowly to a mixture of aq HCl (0.25 M, 660 mL) and ice water (320 mL).The resulting slurry was stirred at 0-10° C. for 0.5 h, filtered, washedwith cold water and dried in vacuo to afford compound 105 as a yellowsolid (22 g, 98%). M.p.:260-265° C. LCMS (m/z): 284.0 [M+1]⁺ ¹H NMR (400MHz, DMSO-d₆): δ 3.77 (t, J=5.2 Hz, 4H), 3.96 (t, J=5.2 Hz, 4H), 8.30(s, 1H), 10.21 (s, 1H).

Step o: (2-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-methyl-amine (Compound 106)

To a solution of compound 105 (20.0 g, 70.4 mmol, 1.0 eq) in methanol(125 mL) was added methylamine solution in methanol (27% v/v, 75 mL,563.2 mmol) under nitrogen atmosphere. The reaction mixture was stirredat room temperature overnight and the solvent was removed in vacuo togive a crude solid product, which was dissolved in methanol (550 mL) andTHF (220 mL) under nitrogen. Sodium borohydride (8 g, 211.2 mmol) wasadded in portions and reaction mixture was stirred at room temperatureovernight. The reaction mixture was evaporated in vacuo and water (300mL) was added. The aqueous mixture was extracted with methylene chlorideand the combined extracts were dried over Na₂SO₄ and concentrated. Theresidue was dissolved in 6M HCl (230 mL) and stirred for 30 min. Theaqueous solution was washed with methylene chloride for several times,and adjusted to pH 9-10 with NaOH (4N). The precipitated solid wascollected by filtration and dried (60° C., 6 h) to give a light yellowsolid (18 g, 85%). M.p.: 240-245° C. LCMS (m/z): 299 [M+1]⁺. ¹H NMR (400MHz, DMSO-d₆): δ 2.32 (s, 3H), 3.74 (t, J=5.2 Hz, 4H), 3.88 (t, J=5.2Hz, 4H), 3.96 (s, 2H), 7.24 (s, 1H).

Step p(a):2-[(2-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-methyl-amino]-pyrimidine-5-carboxylicacid ethyl ester (Compound 107-1)

To a mixture of 106 (10 g, 33.6 mmol) and R-2-1 (6.8 g, 36.4 mmol) inCH₃CN (400 mL) at room temperature was added diisopropylethylamine (220mL, 1.26 mol). The resulting mixture was stirred at room temperatureovernight. The mixture was then evaporated and followed by the additionof methylene chloride (300 mL). The organic phase was washed with water,dried over Na₂SO₄ and concentrated in vacuo to leave a residue. To theresidue was added ethyl acetate and the resulting mixture was stirred atice/water bath temperature for 50 min. The resulting solid was collectedby filtration to give the titled product 107-1 as a white solid (10.6 g,70%). LCMS: 449 [M+1]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 1.30 (t, J=7.2 Hz,3H), 3.25 (s, 3H), 3.71 (t, J=5.2 Hz, 4H), 3.83 (t, J=4.8 Hz, 4H), 4.29(m, 2H), 5.21 (s, 2H), 7.39 (s, 1H), 8.87 (s, 2H).

Step p(b):2-[(2-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-methyl-amino]-pyrimidine-5-carboxylicacid methyl ester (Compound 107-2)

A mixture of compound 106 (25 g, 84 mmol), CH₃CN (500 mL) and R-2-2 (16g, 92 mmol) was stirred at room temperature. Diisopropylethylamine(DIPEA) (500 mL, 2.9 mol) was added. The solution was stirred overnightand evaporated. After methylene chloride (500 mL) was added, the organicphase was washed with water, dried with Na₂SO₄ and concentrated invacuo. To the residue was added ethyl acetate (200 mL) and the mixturewas stirred in ice/water bath for 50 min. The title product wascollected as a white solid (29.4 g, 81%). LCMS (m/z): 435.2 [M+1]⁺.¹HNMR (400 MHz, DMSO-d₆): 3.25 (s, 3H), 3.71 (t, J=5.2 Hz, 4H),3.82-3.84 (m, 7H), 5.21 (s, 2H), 7.39 (s, 1H), 8.87 (s, 2H).

Step q(a):Ethyl-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl) methyl)(methyl)amino)pyrimidine-5-carboxylate (Compound 108-1)

Method A: A mixture of compound 107-1 (12 g, 26.7 mmol), R-3-1 (4.9 g,32 mmol), NaHCO₃ (6.7 g, 80.1 mmol) andbis(triphenylphosphine)palladium(II) chloride (188 mg, 0.267 mmol) in amixed solvents of toluene (80 ml), ethanol (50 ml) and water (10 ml) washeated at 108° C. for 4.5 h under N₂ atmosphere. TLC showed reaction wascomplete. The reaction mixture was then cooled to room temperature andwater (20 ml) was added. The resulting solid was collected by filtrationand was then suspended in ethanol (100 mL). The suspension was stirredat room temperature for 30 minutes and filtered. The collected solid waswashed with ethanol and dried in vacuo to afford titled compound 108-1as a white solid (10 g, 72%).

Method B: A mixture of compound 107-1 (1.5 g, 3.34 mmol), R-3-2 (1.6 g,6.68 mmol), NaHCO₃ (0.84 g,10.0 mmol) andbis(triphenylphosphine)palladium(II) chloride (118 mg, 0.167 mmol) in amixed solvents of toluene (24 ml), ethanol (15 ml),and water (3 ml) washeated at 108° C. under N₂ atmosphere overnight. The reaction mixturewas partitioned between dichloromethane and water. The organic layer wasseparated and was washed with brine, dried over Na₂SO₄, filtered andevaporated in vacuo to give a residue which was purified by columnchromatography eluted with hexanes/ethyl acetate to afford compound108-1 as a white solid (1.7 g, 98%).

m.p.198-202° C. LCMS: 522.30 [M+1]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 1.31(t, J=7.2 Hz, 3H), 3.28 (s, 3H), 3.76 (t, J=4.4 Hz, 4H), 3.93 (t, J=4.4Hz, 4H), 3.94 (s, 3H), 4.30 (q, J=7.2 Hz, 2H), 5.24 (s, 2H), 6.92 (d,J=8.8 Hz, 1H), 7.47 (s, 1H), 8.57 (dd, J=8.8 Hz, 2.0Hz, 1H), 8.88 (s,2H), 9.15 (d, J=2.0 Hz, 1H).

Step q(b):Methyl-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxylate(Compound 108-2)

To a mixture of compound 107-2 (20 g, 46.0 mmol), B-3-1 (9.2 g, 60.2mmol, 1.3 eq.) in dioxane (540 mL) at room temperature was added solidNaHCO₃ (11.6 g, 138.1 mmol, 3 eq.) followed by the addition of water (40mL). The resulting mixture was degassed by passing N₂ through surface ofsolution. Bis(triphenylphosphine) palladium(II) chloride (323 mg, 0.46mmol, 0.01eq.) was then added and the resulting mixture was heated at108° C. for 15 h. TLC and LCMS showed reaction complete. The reactionmixture was filtered through Celite while it was still hot (>90° C.) andwashed with dioxane (70 mL). The filtrate was cooled gradually to roomtemperature and white fine crystals formed during cooling period. Thesuspension was filtered and washed with dioxane (80 mL) to afford thetitled compound 108-2 as a white solid (18 g, 78%). LCMS (m/z): 508.3[M+1]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 3.28 (s, 3H), 3.76 (t, J=4.8 Hz,4H), 3.82 (s, 3H); 3.92 (m, 4H), 3.93 (s, 3H), 5.20 (s, 2H), 6.91 (d,J=8.8 Hz, 1H), 7.47 (s, 1H), 8.57 (dd, J=8.8 Hz, 2.4 Hz, 1H), 8.88 (s,2H), 9.15 (d, J=2.0 Hz, 1H).

Step r:N-Hydroxy-2-((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamide(Compound 1) Preparation of Hydroxylamine Methanol Solution

A mixture of NH₂OH.HCl (80 g, 1.12 mol) in MeOH (400 mL) was heated at60-65° C. for 1 h to form a clear solution. It was then cooled in anice-water bath. To the cold mixture was added a solution of KOH (96 g,1.68 mol) in MeOH (240 mL) dropwise while maintaining the reactiontemperature at 0-10° C. The resulting mixture was stirred at 0° C. for30 minutes and then filtered through a constant pressure funnel filledwith anhydrous Na₂SO₄ (700 g). The filtrate was collected under anice-bath and stored in refrigerator for future use.

Preparation of Compound 1 from Compound 108-1

Compound 108-1 (10 g, 19 mmol) was suspended in the above freshlyprepared hydroxylamine methanol solution (1.79M, 350 ml). To thismixture was added dichloromethane (100 mL). The reaction flask wassealed and the mixture was stirred at room temperature for 5 h before itturned into clear solution. Reaction was stirred for additional 9 h. andwas filtered to remove any insoluble solid. The filtrate was adjusted topH 6-7 with the addition of acetic acid to form solid precipitate. Thesolid was collected by filtration and washed with water and minimumamount of methanol, dried in vacuo at 60° C. for 5 h to afford compound1 as a white solid (9.2 g, 96%). m.p. 177-180° C. LCMS: 509.3 [M+1]⁺. ¹HNMR (400 MHz, DMSO-d₆): δ 3.24 (s, 3H), 3.76 (t, J=5 Hz, 4H), 3.92 (t,J=5 Hz, 4H), 3.92 (s, 3H), 5.20 (s, 2H), 6.90 (d, J=8.8 Hz, 1H), 7.44(s, 1H), 8.57 (dd, J=8.8 Hz, 2.4Hz, 1H), 8.75 (s, 2H), 9.01 (s, 1H),9.14 (d, J=2.0 Hz, 1H), 11.08 (s,1H).

Preparation of Compound 1 from Compound 108-2

To a suspension of compound 108-2 (31 g, 61.1 mmol) in dichloromethane(310 mL) at room temperature was added above freshly preparedhydroxylamine methanol solution (1.79M, 744 ml). The reaction flask wassealed and the reaction mixture was stirred at room temperature for 5 h.The reaction mixture turned into a clear solution. The reaction solutionwas filtered to remove any insoluble solid. To the filtrate was thenadded water (310 mL) and there was no solid formed during the addition.Acetic acid (18.5 mL) was added to adjust pH to 10.20 (continuouslymonitored by pH meter) while stirring. There was no internal temperaturechange during acetic acid addition. The resulting reaction mixture wascontinued to stir for another 4 h. White solid gradually formed. Thesuspension was filtered and washed with minimum amount of methanol (100mL×3). The collected white solid was re-suspended in methanol (620 mL)and water (124 mL) to form a suspension. To the above suspension wasadded additional acetic acid (11 g) to adjust the pH to 5-6. The changeof the solid form was observed. The suspension was continued to stir foranother 2 h and filtered through filter paper and washed with minimumamount of methanol (100 mL×3). The collected white solid was dried inoven (50° C.) for 12 h to afford the title Compound 1 as a white solid(23.6 g, 76.0%). m. p.: 255-259° C. LCMS (m/z): 509.3 [M+1]⁺. ¹H NMR(400 MHz, DMSO-d₆): δ 3.24 (s, 3H), 3.76 (t, J=5.2 Hz, 4H), 3.92 (t,J=5.2 Hz, 4H), 3.92 (s, 3H), 5.20 (s, 2H), 6.91 (d, J=8.4 Hz, 1H), 7.45(s, 1H), 8.57 (dd, J=8.4 Hz, 2.4 Hz, 1H), 8.75 (s, 2H), 9.07 (s, 1H),9.14 (d, J=2.4 Hz, 1H), 11.14 (s,1H).

Example 2 Preparation ofN-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamidemethanesulfonate (Compound 2)

Method A: To a mixture of Compound 1 (300 mg, 0.59 mmol) and MeOH/Et₂O(3/1, 40 mL) was added a solution of methanesulfonic acid (114 mg, 1.18mmol) in MeOH (3 mL) at 0° C. The resulting mixture was stirred at 0° C.for 3 h. The precipitate was collected by filtration and washed withEt₂O to afford Compound 2 as a white solid (260 mg, 73%).

Method B: To a suspension of Compound 1 (1.5 g, 2.95 mmol) indichloromethane/MeOH (40 mL/10 mL) was added methanesulfonic acid (341mg, 3.55 mmol) in 2 mL MeOH at room temperature (15° C.) to form a clearsolution. The reaction mixture was stirred at room temperatureovernight. The reaction mixture was still clear. Ethyl acetate (40 mL)was added to the mixture and continued to stir for 3 h at roomtemperature. The resulting precipitate was collected by filtration toafford Compound 2 as a white solid (1.45 g, 83%).

m.p.: 179-185° C. LCMS: 509.3 [M+1] ¹H NMR (400 MHz, DMSO-d₆): δ 2.35(s, 3H), 3.26 (s, 3H), 3.78 (t, J=9.6 Hz, 4H), 3.95 (s, 3H), 4.03 (t,J=9.2 Hz, 4H), 5.24 (s, 2H), 6.99 (d, J=8.8 Hz, 1H), 7.50 (s, 1H), 8.54(dd, J=8.8 Hz, 2.4 Hz, 1H), 8.76 (s, 2H), 9.12 (d, J=2.4 Hz, 1H), 11.11(br, 1H).

Example 3 Preparation ofN-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamidesodium salt (Compound 3)

To a suspension of Compound 1 (300 mg, 0.59 mmol) in methanol (30 mL) at0° C. was added slowly t-BuONa (85 mg, 0.88 mmol). The resulting mixturewas warmed to room temperature and continued to stir for 2 h. Thereaction was concentrated and the residue was triturated and washed withethanol followed by filtration to afford Compound 3 as a white solid(230 mg, 73%). m.p.: 178-183° C. LCMS: 509.3 [M+1] ¹H NMR (400 MHz,DMSO-d₆): δ 3.17 (s, 3H), 3.75 (s, 4H), 3.92 (s, 7H), 5.16 (s, 2H), 6.90(d, J=8.4 Hz, 1H), 7.42 (s, 1H), 8.57 (d, J=8.0 Hz, 1H), 8.65 (s, 2H),9.14 (s, 1H).

Example 4 Preparation ofN-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamidepotassium salt (Compound 4)

To a mixture of Compound 1 (400 mg, 0.78 mmol) in methanol (50 mL) wasadded t-BuOK (132 mg, 1.17 mmol) at 0° C. under N₂. The mixture wasstirred at 0° C. for 1 h and continued to stir at room temperature for1.5 h. The insoluble solid was removed by filtration and the filtratewas cooled to −20° C. Et₂O (100 mL) was added to the filtrate. Theresulting mixture was stirred at −20° C. for 1 h. Hexanes (70 mL) wasadded and the mixture was continued to stir at −20° C. for 2 h. Thesolid was collected by filtration and dried in vacuo to afford Compound4 as a white solid (150 mg, 35%). m.p.: 174-179° C. LCMS: 509.3[M+1]⁺.¹H NMR (400 MHz, DMSO-d₆): δ 3.16 (s, 3H), 3.74-3.76 (m, 4H), 3.90-3.93(m, 7H), 5.15 (s, 2H), 6.90 (d, J=8.4Hz, 1H), 7.43 (s, 1H), 8.39 (br,1H), 8.58 (d, J=8.8Hz, 1H), 8.62 (s, 2H), 9.15 (s, 1H).

Example 5 Preparation ofN-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamidecholine salt (Compound 5)

To a solution of Compound 1 (200 mg, 0.39 mmol) in DCM/MeOH (60 mL/12mL) was added choline hydroxide (106 mg, 0.39 mmol, 45% in MeOH). Themixture was stirred at room temperature for 2 h and was thenconcentrated to remove ˜30 mL of the solvent. Ethyl acetate (60 mL) wasadded and the mixture was stirred at room temperature for 2 h. After asmall amount of precipitation occurred, the mixture was concentrated toremove ˜40 mL of the solvent and additional ethyl acetate (60 mL) wasadded. The mixture was stirred at room temperature for 2 h and filteredto afford Compound 5 as a white solid (180 mg, 76%). m.p.: 181-185° C.LCMS: 509.3[M+1]⁺. ¹H NMR (400MHz, DMSO-d₆): δ 3.11 (s, 9H), 3.17 (s,3H), 3.40 (t, J=4.8 Hz, 2H), 3.75 (t, J=4.8 Hz, 4H), 3.84 (br, 2H),3.90-3.93 (m, 7H), 5.15 (s, 2H), 6.89 (d, J=8.8 Hz, 1H), 7.41 (s, 1H),8.57 (dd, J=8.8 Hz, 2.4 Hz, 1H), 8.64 (s, 2H), 9.14 (d, J=2.0 Hz, 1H).

Example 6 Preparation ofN-hydroxy-2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamidesulfate (Compound 6)

To a suspension of Compound 1 (200 mg, 0.39 mmol) in DCM/MeOH (30 mL/7.5mL) was added sulfuric acid (77 mg, 0.79 mmol, in 1 mL MeOH) to form aclear solution. The reaction mixture was stirred at room temperatureovernight. The precipitation occurred and tert-butyl methyl ether (60mL) was then added. The resulting mixture was continued to stir for 1 hat room temperature. The solid was collected by filtration to affordCompound 6 as a white solid (180 mg, 76%). M.p.: 243-246° C. LCMS: 509.3[M+1]⁺. ¹H NMR (400 MHz, DMSO-d₆): δ 3.26 (s , 3H), 3.78 (t, J =4.8 Hz,4H), 3.96 (s, 3H), 4.03 (t, J =4.4 Hz, 4H), 5.24 (s, 3H), 6.98 (d, J=8.4 Hz, 1H), 7.50 (s , 1H), 8.54 (dd, J =8.8 Hz, 2.4 Hz, 1H), 8.76 (s,2H), 9.12 (d, J =2.0 Hz, 1H) , 11.06 (br , 1H).

Example 7 PI3 Kinase Activity Assay

The following assays were used to determine the ability of Compound 1 toinhibit various isoforms and mutants of PI3K.

PI3Kα

PI3Kα activity was measured using ADP-Glo luminesceni kinase assay.P13Kα, a complex of N-terminal GST-tagged recombinant full-length humanp110α and untagged recombinant full length human p85α were coexpressedin a Baculovirus infected Sf9 cell expression system. (GenBank AccessionNo. for p110α, U79143; for p85α, XM_043865). The proteins were purifiedby one-step affinity chromatography using glutathione-agarose. Acompetition assay was performed to measure the amount of ADP generatedfrom ATP in the presence of purified recombinant PI3Kα (p110α/p85α) andPIP2. PI3Kα was incubated with 20 μM PIP2 substrate in the reactionbuffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 3 uMNaorthovanadate, 1 mM DTT, 10 μM ultra pure ATP and 0.5% DMSO) for 30minutes at 30° C. The ADP generated in the reaction was then measured bythe ADP-Glo Assay. The assay was performed in two steps; first an equalvolume of ADP-GLOTM Reagent (Promega) was added to terminate the kinasereaction and deplete the remaining ATP. In the second step, the KinaseDetection Reagent was added, which simultaneously converts ADP to ATP.The newly synthesized ATP was measured using coupledluciferase/luciferin reaction. The IC₅₀ determined for Compound 1 inthis assay was less than 100 nM.

The ability of Compound 1 to inhibit the PI3Ka mutants H1047R and E545Kwas also determined using the general procdure described above. The IC₅₀determined for both mutants was less than 100 nm.

PI3Kβ

Activity of PI3Kβ was measured using time-resolved fluorescenceresonance energy transfer (TR-FRET) assay utilizing homogenous timeresolved fluorescence (HTRF) technology. P23Kβ, a complex of N-terminalhistidine-tagged recombinant full-length human p110β and untaggedrecombinant full length human p85α were coexpressed in a Baculovirusinfected Sf21 cell expression system. (GenBank Accession No. for p110β,NM_006219; for p85α, XM_043865). The proteins are purified by one-stepaffinity chromatography using glutathione-agarose. A competition assaywas performed to measure the amount of PIP3 generated from PIP2 in thepresence of purified recombinant PI3Kbeta (p110β/p85α). PI3Kβ wasincubated with 10 μM PIP2 substrate in the reaction buffer (20 mM HEPES,pH 7.5, 10 mM NaCl, 4 mM MgCl₂, 2 mM DTT, 10 μM ATP and 1% DMSO) for 30minutes at 30° C. The reaction product was then mixed with a PIP3detector protein, europium-labeled antibody, biotin-labeled PIP3 probeand allophycocyanin-labeled Streptavidin. A sensor complex is formed togenerate a stable TR-FRET signal in the reaction mixture. This signalintensity decrease as biotin-labeled probe binding to the PIP3 detectoris displaced by PIP3 produced by enzymatic activity and the amount ofunbound biotin-labeled PIP3 probe in the mixture increases. TR-FRETsignal was determined using microplate reader with backgroundsubtraction.

The IC₅₀ determined for Compound 1 in this assay was between 100 and1000 nM.

PI3Kδ

Activity of PI3Kδ was measured using fluorescence polarization assay.P13Kδ, a complex of N-terminal histidine-tagged recombinant full-lengthhuman p110δ and untagged recombinant full length human p85α werecoexpressed in a Baculovirus infected Sf9 cell expression system.(GenBank Accession No. for p110δ, NM_005026). The proteins are purifiedby one-step affinity chromatography using glutathione-agarose. Acompetition assay was performed to measure the amount of PIP3 generatedfrom PIP2 in the presence of purified recombinant PI3Kδ (p110δ/p85α).PI3Kδ was incubated with 10 μM PIP2 substrate in the reaction buffer (20mM HEPES (pH 7.5), 10 mM NaCl, 4 mM MgCl₂, 2 mM DTT, 10 μM ATP and 1%DMSO) for 1 hour at 30° C. The reaction product was then mixed with aPIP3 detector protein and the fluorescent PIP3 probe. Polarization (mP)values decrease as fluorescent probe binding to the PIP3 detector isdisplaced by PIP3 produced by enzymatic activity and the amount ofunbound fluorescent probe in the mixture increases. Polarization degrees(mP) value was determined using microplate reader with backgroundsubtraction.

The IC₅₀ determined for Compound 1 in this assay was less than 100 nM.

PI3Kγ

Activity of PI3Kγ was measured using time-resolved fluorescenceresonance energy transfer (TR-FRET) assay utilizing homogenous timeresolved fluorescence (HTRF) technology. N-terminal histidine taggedhuman P13Kδ was expressed in a Baculovirus infected Sf9 cell expressionsystem. (GenBank Accession AF327656). The proteins are purified byone-step affinity chromatography using glutathione-agarose. Acompetition assay was performed to measure the amount of PIP3 generatedfrom PIP2 in the presence of purified recombinant PI3Kγ (p120γ). PI3Kγ(2 nM) was incubated with 10 μM PIP2 substrate in the reaction buffer(20 mM HEPES, pH 7.5, 10 mM NaCl, 4 mM MgCl₂, 2 mM DTT, 10 μM ATP and 1%DMSO) for 30 minutes at 30° C. The reaction product was then mixed witha PIP3 detector protein, europium-labeled antibody, biotin-labeled PIP3probe and allophycocyanin-labeled Streptavidin. A sensor complex isformed to generate a stable TR-FRET signal in the reaction mixture. Thissignal intensity decrease as biotin-labeled probe binding to the PIP3detector is displaced by PIP3 produced by enzymatic activity and theamount of unbound biotin-labeled PIP3 probe in the mixture increases.TR-FRET signal was determined using microplate reader with backgroundsubtraction.

The IC₅₀ determined for Compound 1 in this assay was between 100 and1000 nM.

Example 8 HDAC Activity Assay

HDAC inhibitory activity was assessed using the Biomol Color de Lyssystem (AK-500, Biomol, Plymouth Meeting, Pa.). Briefly, HeLa cellnuclear extracts were used as a source of HDACs. Differentconcentrations of test compounds were serially diluted indimethylsulfoxide (DMSO) and added to HeLa cell nuclear extracts in thepresence of a colorimetric artificial substrate. Final assay conditioncontained 50 mM Tris/Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl₂.Reactions were carried in room temperature (25° C.) for 1 hour beforeaddition of developer for termination. Relative enzyme activity wasmeasured in the WALLAC Victor II 1420 microplate reader as fluorescenceintensity (excitation: 350-380 nm; emission: 440-460 nm). Data wereanalyzed using GraphPad Prism (v4.0a) with a sigmoidal dose responsecurve fitting for IC₅₀ calculation. The IC₅₀ determined for Compound 1in this assay was less than 100 nM.

The activities of Compound 1 against HDAC isotypes were also determined.HDAC specificity assays were performed at BPS Bioscience (San Diego,Calif.), following their standard operating procedure. Briefly, purifiedflag- (human HDAC-1), NCOR2- (human HDAC3), GST- (human HDAC4, 6, 7, 10and 11) or His- (human HDAC 2, 5, 8 and 9) tagged enzymes were expressedin Sf9 insect cells and purified before use. The substrate used forHDAC1, 2, 3, 6, 7, 8, 9 and 11 was HDAC Substrate 3 developed by BPSBioscience. For other HDAC enzymes, HDAC Class 2a substrate was used.All enzymatic reactions were conducted in duplicate at 37° C. for 30minutes, except HDAC11 enzyme assay, which was conducted at roomtemperature for 3 hours.

The table below sets forth the results for each of HDACs 1-11, with IC50values provided as follows: I>1000 nM; 100 nM<II<1000 nM; 10 nM<III<100nM; IV<10 nM.

HDAC 1 2 3 8 4 5 6 7 9 10 11 IC₅₀ IV IV IV II II II III II II IV IV

Example 9 Cell Proliferation Assay

Human cancer cell lines were purchased from American Type CultureCollection (Manassas, Va.) and plated at 5,000 to 10,000 per well in96-well flat-bottomed plates with culture medium, as suggested by theprovider. The cells were then incubated with compounds at variousconcentrations for 72 hours in culture medium supplemented with 0.5%(v/v) fetal bovine serum (FBS). Growth inhibition was accessed byadenosine triphosphate (ATP) content assay using Promega CellTiter-Glokit. Promega CellTiter-Glo kit is an ATP monitoring system based onfirefly luciferase. Briefly, 16 μl of mammalian cell lysis and substratesolution was added to 84 μl of culture medium per well to lyse the cellsand stabilize the ATP. The mixture was shaken and incubated for 30minutes and subsequently the luminescence was measured. IC₅₀ values werecalculated using PRISM software (GraphPad Software) with sigmoidaldose-response curve fitting.

Table 1 shows the antiproliferative activity in these cell-based assaysof Compound 1 and reference compounds SAHA, GDC-0941 and the combinationof SAHA and GDC-0941. In these assays, the following grading was used:I>10,000 nM, 10,000 nM>II>1000 nM, 1000 nM>III>100 nM, 100 nM>IV>10 nM,and V <10 nm for IC₅₀.

TABLE 1 Cancer Type GDC- SAHA/ Cell Line SAHA 0941 GDC-0941 Cmpd 1 ColonWiDr II I III IV HCT116 II II III V SW403 II I II V SW620 II I III VSWI-116 II I II V T-84 II II III IV NSCLC H358 II II II V H292 II II IIIV H2122 II II III V H460 I I II IV A549 II II II IV Calu6 II I II IVPancreas MiaPaca2 II I II IV CaPan2 II I III IV CFPAC-1 II I II IVPANC-1 II II II IV SW1990 II I II V Breast HCC1500 II I II V HCC1806 III II IV MDA-MB-231 II I II IV SKBr3 II I II IV BT474 II III III VMDA-MB-361 II III IV V UACC-893 II II III IV MDA-MB-453 III III III VMCF-7 II III III V T47D II I III IV ZR-75-1 II III II IV MDA-MB-468 IIIII II IV ALL MOLT-4 III III III V SUP-B15 III II III V AML HL-60 IIIIII III V U937 III II III V THP-1 I II III IV MV-4-11 III II III VB-Cell Pfeiffer II III III V Lymphoma Raji II I II IV RL III II III VDOHH2 III IV IV V Granta 519 II I III V Su-DHL4 II III III V Daudi II IIII IV T-Cell HH III III III V Lymphoma MJ III I III V HuT78 IV III IV VCML K562 II II III IV MEG-01 II I II V Multiple RPMI-8226 II I III VMyeloma OPM-2 III IV III V ARH77 II I III V

Example 10 Formulations of Compound 1

a. Compound 1 in 30% Captisol (10 mg/mL):

To a vial containing compound 1 (10 mg) was added 30% Captisol (0.937ml). The mixture was sonicated for 2 min. To the mixture was addedsodium hydroxide (1 N, 39.3 2 eq.) and sonicated/vortexed to give aclear solution (pH =12). The solution was then adjusted to pH=10 withhydrochloric acid (1 N, 23.6 μl, 1.2 eq.).

b. Compound 1 in 30% Captisol (7.5 mg/mL):

To a vial containing compound 1 (7.5 mg) was added 30% Captisol (0.941ml). The mixture was sonicated for 2 min. To the mixture was addedsodium hydroxide (1 N, 29.5 μl, 2 eq.) and sonicated/vortexed to give aclear solution (pH=12). The solution was then adjusted to pH =5 withhydrochloric acid (1 N, 29.5 μl, 2 eq.).

c. Compound 1 in C10/PEG1450/PEG400 (5 mg/mL):

To a vial containing compound 1 (5 mg), sodium decanoate (20 mg), PEG400(40 μl), and PEG1450 (40 mg) was added H₂O (0.88 ml) and NaOH (1 N, 24.6μl, 2.5 eq.). The mixture was sonicated and vortexed to give a clearsolution which was then adjusted to pH=10 with HCl (1 N, 7.4 μl, 0.75eq.).

Example 11 Pharmacokinetics and Pharmacodynamics Studies inTumor-Bearing Mice Nude Mice Bearing H2122 Tumors

Nude mice bearing H2122 (human non-small cell lung cancer cell line)xenograft tumors were used for pharmacokinetics studies. Compound 1 wasformulated in water with sodium decanoate and PEG400 (5 mg/ml) and wasadministered orally (PO) via gavage to each animal at a dose of 50mg/kg. At various time points following compound administration, threemice per time point were euthanized with CO₂, and blood and tumortissues were collected. Blood was collected into tubes containing sodiumheparin. The plasma was separated via centrifugation. Plasma and tissueswere stored at −80° C. for later analysis. A PE Sciex API-3000 LC-MS/MSsystem (Applied Biosystems, Inc., Foster City, Calif.) was used toanalyze compound concentrations in plasma and tumor tissues.

The results of this study are summarized in FIG. 1 and Table 2, below.FIG. 1 is a graph of Compound 1 concentration in plasma and tumor tissueversus time following oral administration. The results show thatCompound 1 preferentially accumulates in tumor tissue. This is supportedby the results set forth in Table 3, which show a significantly longerhalf-life of Compound 1 in tumor tissue than in plasma as well assignificantly greater exposure of tumor tissue to Compound 1 (AUC).

TABLE 2 Parameter Plasma Tumor Half-life (Hours) 5.9 10.1 C_(max)(ng/mL) 186 154 Area under the Curve 478 2126 (ng/mL * hr)Bioavailability (%) 7.8 14.8SCID Mice bearing Daudi Tumors

Daudi (non-Hodgkin's lymphoma cell line) cells were implanted intofemale Scid (severe complex immune-deficient) mice. Followingestablishment of tumors, animals were dosed by oral gavage with 25, 50or 100 mg/kg Compound 1, formulated in 30% Captisol, pH 10, at aconcentration of 1.875, 3.75 or 7.5 mg/mL, respectively.

At various time points following compound administration, three mice pertime point were euthanized with CO₂, and blood and tumor tissues werecollected. Blood was collected into tubes containing sodium heparin. Theplasma was separated via centrifugation. Plasma and tissues were storedat −80° C. for later analysis. A PE Sciex API-3000 LC-MS/MS system(Applied Biosystems, Inc., Foster City, Calif.) was used to analyzecompound concentrations in plasma.

The results of this study are summarized in FIGS. 2A, 2B and 2C andTable 3, below. FIG. 2A is a graph of Compound 1 concentration in plasmaversus time following oral administration and shows a dose-dependentexposure to the compound. FIG. 2B is a graph of Compound 1 concentrationin tumor tissue versus time following oral administration.

The results show that Compound 1 preferentially accumulates in tumortissue in a dose dependent manner. Plasma and tumor concentrationsfollowing the 100 mg/kg dose are compared in FIG. 2C, which shows thattumor tissue preferentially takes up Compound 1. This is supported bythe results set forth in Table 3, which show a significantly longerhalf-life of Compound 1 in tumor tissue than in plasma as well assignificantly greater exposure of tumor tissue to Compound 1 (AUC).

TABLE 3 Parameter Plasma Tumor Half-life (Hours) 7.73 12.62 C_(max)(ng/mL) 2285.39 1044.7 Area under the Curve 1899.26 3973.56 (ng/mL * hr)T_(max) (hr) 0.24 0.10

Pharmacodynamics

Tumors were collected for PD evaluation following treatment with asingle dose of Compound 1 at 25 mg/Kg, 50 mg/kg and 100mg/kg. Proteinwas extracted from tumor tissues using a Tissuelyser (Qiagen, Valencia,Calif.) according to the manufacturer's instructions. 30 ug of proteinwas routinely used for WB analysis as described above. Cell lysates wereresolved on NuPAGE Novex 4-12% Bis-Tris gels (Invitrogen) andtransferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules,Calif.). The blots were probed with various primary antibodies overnightat 4° C. GAPDH (glyceraldehyde 3-phosphate dehydrogenase, 1:30,000,Abcam, Cambridge, Mass.) was used as an internal control for each assay.Membranes were then incubated with infrared labeled secondary antibodies(1:10000) conjugated-IR Dye-800 (Rockland Immunochemicals, Inc.Gilbertsville, Pa.) or conjugated-Alexa 680 (Invitrogen). Membranes wereimaged with the Odyssey Infrared Imaging System (Li-Cor Biotechnology,Lincoln, Nebr.).

The results of this study are set forth in FIG. 3, which presentsWestern blots of tumor tissue extracts from the three dose groups. Theseresults show that Compound 1 inhibits the PI3K-AKT-mTOR pathway,supresses the RAF-MEK-ERK pathways, downregulates RTK protein levels andup-regulates tumor suppressor p53 and p21 levels.

Example 12 Pharmacokinetic Study in Dogs

A pharmacokinetic study of Compound 1 in beagle dogs was also conductedusing iv administration at 5 mg/kg in water with sodium decanoate/PEG400(5 mg/ml) and oral administration at 5 mg/kg with sodiumdecanoate/PEG4000/PEG1450 (pH 10) in enteric capsules. Plasma wascollected at various time points and analyzed for Compound 1concentration by LC-MS/MS. The results of the study are shown in FIG. 4and Table 4, below. FIG. 4 is a graph of plasma concentration versustime for both oral and iv dosing. Significant plasma levels of Compound1 are achieved via oral dosing.

TABLE 4 Parameter IV PO Capsule Half-life (Hours) 1.85 4.88 C_(max)(ng/mL) 6156.16 312.1 Area under the Curve 2977.47 450.4 (ng/mL * hr)Bioavailability (%) 15.1

Example 13 Pharmacokinetic Study in Rats

The purpose of this study was to determine the plasma pharmacokineticsof Compound 1 in male Sprague-Dawley rats following oral administrationof Compound 1.

Compound 1 was dissolved in 30% Captisol in water to yield a nominalconcentration of 10 mg/mL (pH=10) for oral administration. The resultingclear yellow solution was stored at room temperature until picked up fordosing.

Three male Sprague-Dawley rats from Charles River Laboratories were usedin this study. High fat diet (VHFD, D12492i) from Research Diets Inc.were provided ad libitum throughout the in-life portion of the study.Compound 1 was administered via a single oral (PO) gavage dose at 20mg/kg.

Blood samples (approximate volume 150 μL) were collected tail vein at0.25, 0.5, 1, 3, 6, and 24 hours postdose. Blood samples were placedinto tubes containing sodium heparin and centrifuged at 8000 rpm for 6minutes at 4° C. to separate plasma from the samples. Followingcentrifugation, the resulting plasma was transferred to clean tubes andstored frozen at −80° C. pending bioanalysis.

The concentrations of Compound 1 and its primary metabolite in theplasma samples were determined using a PE Sciex API-3000 LC-MS/MS system(PE-Sciex., Foster City, Calif.).

The pharmacokinetic parameters were determined from meanconcentration-time data in the test subjects. A compartmental modelingof WINNONILIN® Professional 5.2. was used to calculate parameters. Anyconcentrations that were below the limit of quantitation (lower limit ofquantitation=1 ng/mL) were omitted from the calculation of parameters inindividual animals.

Following oral administration of Compound 1, the mean values of C_(max).and T_(max) for Compound 1 were 39.5 μg/L and 0.1 hr, respectively. Themean value of AUC(_(0-∞)) was 163.6 μg/L*hr. The value of half-life(T_(1/2)) was 11.7 hr.

Example 14 Evaluation of Compound 1 in Xenograft Tumor Models

A. SU-DHL4, H2122, Daudi and OPM2 Xenograft Tumor Models

SU-DHL4 (diffuse large B-cell lymphoma cell line), H2122 (human NSCLCcell line), Daudi (non-Hodgkin's lymphoma cell line), and OPM2 (multiplemyeloma tumor cell line) cells were implanted into either nude or Scid(severe complex immune-deficient) mice. Following establishment oftumors, animals with sufficient tumor size were randomly assigned intoactive (Compound 1) and control (vehicle) groups. Compound 1 wasformulated for oral administration as in Example 7(b), and delivered byoral gavage based on the body weight of each individual animal. Thecontrol groups were treated with vehicle using the same dosing scheduleas the corresponding active group.

The H2122 tumor group (nude mice) received Compound 1 at doses of 75mg/kg twice a day initially and then 50 mg/Kg twice a day from Day-11for five days per week due to body weight loss at 75 mg/Kg. In onestudy, the Daudi tumor group (Scid mice) received Compound 1 at doses of25, 50 or 100 mg/Kg five day per week. In another study, the Daudi tumorgroup was dosed at 50 mg/Kg twice a day for five days per week. Inanother study, the efficacy of orally administered Compound 1 in theDaudi tumor model was compared to oral GDC-0941 and oral vorinostat,both individually and in combination. The OPM2 tumor group receivedCompound 1 at doses of 50 mg/kg twice a day for five days per week. TheSU-DHL4 tumor group was dosed at 100 mg/Kg orally or 50 mg/Kgintravenously.

Tumors were measured during the study period with an electronic caliper,and body weights were measured twice a week. The following formula wasused to calculate the tumor volume:

Tumor volume=(length×width²)/2

Percentage of tumor volume change was used to describe compound activityover the treatment period.

The results of these studies are summarized in FIGS. 5A to 5C and 9 to12, which show tumor size versus time for active and control groups foreach of the tumor types. FIGS. 5A, 5B, 5C and 12 show that Compound 1 isefficacious in the H2122, Daudi and OPM2 tumor models. As set forth inFIG. 9, Compound 1 inhibited Daudi tumor growth in a dose-dependentmanner. FIG. 10 compares the antitumor activity of Compound 1 at 100mg/Kg in the Daudi model with either GDC-0941 or vorinostat alone or incombination. The indicated doses are the maximum tolerated dose (MTD) ofeach treatment, and the pretreatment tumor size was 157±65 mm³(mean±SE). The data indicate that Compound 1 is more efficacious thanvorinostat, GDC-0941, or a combination of both. Finally, Compound 1strongly inhibited tumor growth in the SU-DHL4 diffuse large B-celllymphoma xenograft model following intravenous (IV) administration at 50mg/kg or orally (PO) at 100 mg/kg (FIG. 11). The pretreatment tumor sizewas 147±21 mm³.

MM1S Xenograft Model

Female SCID/Beige mice at age 4 weeks were housed in ventilatedmicro-isolator cages (INNOCAGE®IVC, Innovive Inc., San Diego, Calif.) ina controlled climate, fed with sterile high-fat diet (Problab-RMH 2000)ad libitum and provided with sterilized water. All housing and suppliesfor SCID/Beige mice were sterilized by autoclaving before use. Mice wereinspected daily including weekends/holidays by trained animal facilitypersonnel and investigators. All animal procedures were performed understerile conditions within a biosafety cabinet (for injections) orlaminar flow hood (for animal husbandry and non-invasive procedures).

MM1S human MM cells (Goldman-Leikin R E, et al., J Lab Clin Invest.1980; 113:335-345) were originally obtained from peripheral blood of amultiple myeloma patient. Cryopreserved cells were thawed in a 37° C.water bath and cultured in RPMI medium plus 10% Fetal Bovine Serum (FBS)in a tissue culture incubator at 5% CO₂. Cells were sent to outsidevendors for contaminants and rodent pathogen screening intended to ruleout contamination by mycoplasma (by PCR) and/or virus (by MAP test,Mouse Antibody Production). When the cells in culture were enough forimplantation, they washed with serum free Hank's balanced salt solution(HBSS). Finally the cells were diluted in HBSS for implantation. Onlysingle-cell suspensions of greater than 90% viability (by trypan blueexclusion) were used for injection and 20 million cells per animalsuspended in 0.2 ml HBSS were injected subcutaneously in the right hindflank region of the mouse after a minimum 7 day acclimation period,using a 1 CC syringe with a 26G hypodermic needle, taking care to avoidblood vessels. Successful implantation was indicated by the formation ofa round, raised mass under the skin. The implanted mice were monitoredfor general health and tumor development daily.

Tumors were detectable about two weeks following implantation. Tumorsize was measured with a caliper. The following formula was used tocalculate the tumor volume:

Tumor volume=(length×width²)/2

Three weeks after tumor implantation, tumors reached an average of194.6±37.9 mm³. Animals with acceptable tumor size and shape wererandomly assigned into two groups of eight animals each, using sortingsoftware, one vehicle control and one treatment group.

Compound 1 was formulated and dosed as follows: 7.5 mg/ml was dissolvedin 30% Captisol with 2 molar equivalents of NaOH and HCl each and dosedby oral gavage everyday five times per week based on body weight of eachmouse. The control group was dosed with vehicle (30% Captisol) usingsame dosing paradigm.

During each animal study, tumors were measured with calipers, tumor sizedetermined using the above mentioned formula, and tumor size changes inpercentage calculated. Mouse body weights were measured with a scaletwice per week. Studies were continued until either: a) thepredetermined end date indicated in the study design; or b) the onset ofhealth problems, whichever occurred first. In addition, the followingtumor-related parameters warranted provision of euthanasia: (1) tumorburden exceeding 2500 mm³ and/or (2) loss of 20% of starting bodyweight. In addition to the determination of tumor size changes, the lasttumor measurement was used to generate the tumor weight change ratio(T/C value), a standard metric developed by the National CancerInstitute (NCI) for xenograft tumor evaluation T/C values werecalculated using the following formula: % T/C=100×ΔT/ΔC if ΔT>0. Incases where tumor regression occurred, however, the following formulawas used: % T/T₀=100×ΔT/T0 if ΔT<0.

The treatment period was 15 days. Tumor sizes and body weights weremeasured again on the last day of the study.

As shown in FIG. 13, Compound 1 single agent inhibited tumor growth inthe MIVI1S subcutaneous tumor model. The T/C values are calculated to be27.37% (p<0.0001, ANOVA) based on day 14. No body weight loss or otherside effects were observed for the Compound 1 single agent treatmentgroup.

MM1R Xenograft Model

Female SCID/Beige mice at age 4 weeks were housed in ventilatedmicro-isolator cages (INNOCAGE®IVC, Innovive Inc., San Diego, Calif.) ina controlled climate, fed with sterile high-fat diet (Problab-RMH 2000)ad libitum and provided with sterilized water. All housing and suppliesfor SCID/Beige mice were sterilized by autoclaving before use. Mice wereinspected daily including weekends/holidays by trained animal facilitypersonnel and investigators. All animal procedures were performed understerile conditions within a biosafety cabinet (for injections) orlaminar flow hood (for animal husbandry and non-invasive procedures).

MM1R human MM cells were originally obtained from peripheral blood of amultiple myeloma patient (Goldman-Leikin R E, et al., J lab Clin Invest.1980, 113:335-345). Cryopreserved cells were thawed in a 37° C. waterbath and cultured in RPMI medium plus 10% Fetal Bovine Serum (FBS) in atissue culture incubator at 5% CO₂. Cells were sent to outside vendorsfor contaminants and rodent pathogen screening intended to rule outcontamination by mycoplasma (by PCR) and/or virus (by MAP test, MouseAntibody Production). When the cells in culture were enough forimplantation, they washed with serum free Hank's balanced salt solution(HBSS). Finally the cells were diluted in HBSS for implantation. Onlysingle-cell suspensions of greater than 90% viability (by trypan blueexclusion) were used for injection and 15 million cells per animalsuspended in 0.1 ml HBSS were injected subcutaneously in the right hindflank region of the mouse after a minimum 7 day acclimation period,using a 1 CC syringe with a 26G hypodermic needle, taking care to avoidblood vessels. Successful implantation was indicated by the formation ofa round, raised mass under the skin. The implanted mice were monitoredfor general health and tumor development daily.

Tumors were detectable about two weeks following implantation. Tumorsize was measured with a caliper. The following formula was used tocalculate the tumor volume:

Tumor volume=(length×width²)/2

Three weeks after tumor implantation, tumor reached an average of131.7±28.7 mm³. Animals with acceptable tumor size and shape wererandomly assigned into two groups of eight animals each, using sortingsoftware, one vehicle control and one treatment group.

Compound 1 was formulated and dosed as follows: 7.5 mg/ml was dissolvedin 30% Captisol with 2 molar equivalents of NaOH and HCl each and dosedby oral gavage everyday five times per week based on body weight of eachmouse. The control group was dosed with vehicle (30% Captisol) usingsame dosing paradigm.

During each animal study, tumors were measured with calipers, tumor sizedetermined using the above mentioned formula, and tumor size changes inpercentage calculated. Mouse body weights were measured with a scaletwice per week. Studies were continued until either: a) thepredetermined end date indicated in the study design; or b) the onset ofhealth problems, whichever occurred first. In addition, the followingtumor-related parameters warranted provision of euthanasia: (1) tumorburden exceeding 2500 mm³ and/or (2) loss of 20% of starting bodyweight. In addition to the determination of tumor size changes, the lasttumor measurement was used to generate the tumor weight change ratio(T/C value), a standard metric developed by the National CancerInstitute (NCI) for xenograft tumor evaluation. T/C values werecalculated using the following formula: % T/C=100×ΔT/ΔC if ΔT>0. Incases where tumor regression occurred, however, the following formulawas used: % T/T₀=100×ΔT/T0 if ΔT<0.

The treatment period was 18 days. Tumor sizes and body weights weremeasured again on the last day of the study.

As shown in FIG. 14, Compound 1 single agent inhibited tumor growth inthe MM1R subcutaneous tumor model. The T/C values are calculated to be21.15% (p<0.0001, ANOVA) based on day-17. No body weight loss or otherside effects were observed for the Compound 1 single agent treatmentgroup.

Example 15 Effect of Compound 1 on Circulating Lymphocytes

A study examining the effect of Compound 1 on circulating T and Blymphocytes was conducted in CD1 wild type mice. Five mice were treatedwith Compound 1 formulated as in Example 8(b) (5 mg/mL) at 100 mg/kgorally for five consecutive days. Another 5 mice were treated withvehicle. Blood was collected at various time points (includingpre-dosing, during dosing and post-dosing) from the mandibular vein.Blood was analyzed with a flow cytometer for T and B cellquantification.

The effect of Compound 1 on T and B lymphocyte levels in lymphoidorgans, spleen and lymph nodes, was also evaluated. Mice were treatedwith Compound 1 orally at 100 mg/kg for five consecutive days. Theanimals were sacrificed, and the lymphoid organs were collected. Cellswere physically dissociated from the tisues and analyzed with a flowcytometer. Anti-CD3 and -CD 19 antibodies were used to stain T and Bcells, respectively.

The results of these studies are shown in FIG. 6, a graph showing bloodlymphocyte levels over time. Compound 1 shows a significant reversiblereduction in the blood levels of both T and B lymphocytes compared tocontrol. A similar effect is seen in lymphocyte levels in the spleen andlymph nodes. Both of these organs show a significant reduction in both Tand B lymphocytes following dosing with Compound 1 compared to controls.

Example 16 Effect of Compound 1 on Hematopoietic Cells in Bone Marrow

Bone marrow was also removed from the mice sacrificed in Example 12.Bone marrow content were collected from the mice long bones and analyzedwith flow cytometer. Various markers for progenitor or maturelymphocytes were used. The results showed that treatment with Compound1, while causing a decrease in peripheral T and B lymphocyte counts,induced a compensatory increase in marrow lymphocyte progenitor cellscompared to controls.

Example 17 Mini-Salmonella/Mammalian-Microsome Reverse Mutation Assay

This study was conducted to evaluate the ability of Compound 1 to inducereverse mutations either in the presence or absence of mammalianmicrosomal enzyme (S9-mix) at the histidine locus in the genome of 2strains of Salmonella typhimurium (TA98 and TA100).

The tester strains used in the mutagenicity assay were Salmonellatyphimurium tester strains TA98 (for detecting frame-shift reversemutation) and TA100 (for detecting point reverse mutation). The assaywas conducted in both the presence and absence of S9 mixture along withconcurrent vehicle (DMSO, 20 μl/well) and positive controls in duplicateusing E-well plates. Five concentrations with 2× succeeding dilutionsranging from 1000 to 62.5 μg/well (equivalent to 5000 to 312.5 μg/platein standard Ames assay) were tested for each of the compounds. Afterincubation at 37° C. for 48-72 hours, plates were observed for compoundinsolubility and cytotoxicity, and scanned to count revertants colonies.A reproducible two-fold increase (>2× of vehicle control) of revertantcolonies over the daily average control value is considered a positiveresponse of gene mutation for each strain.

Compound 1 was dissolved in dimethyl sulfoxide (DMSO), which also servedas the negative (vehicle) control. 2-nitrofluorene and sodium azideserved as the positive controls in the absence of S9 for TA98 and TA100respectively. 2-Aminoanthracene served as the positive controls in thepresence of S9 for TA98 and TA100.

Results

Compound 1 formed a maroon solution when dissolved in DMSO at aconcentration of 50 mg/ml, which was the most concentrated stocksolution. The test article remained a light maroon to colorless solutionin all 2× succeeding dilutions down to 3.125 mg/ml. The precipitation ofthe test article was observed when the test article and soft agar weremixed together at the concentration of 250 μg/well and above. After48-72 hours incubation, test article precipitation was seen slightlyunder dissecting microscopy at 250 μg/well with TA98 and TA100 in theabsence of S9 mix only, test article precipitation and minor reductionof background lawn were seen slightly to moderately at 500 and 1000μg/well with TA98 and TA100 in the presence and absence of S9 mix. Therewas no evidence of a significant increase in the mean number ofrevertant colonies compared to the average control when tested in thepresence and absence of S9 mix with strains TA98 and TA100 (Table 5).

Results from the current study showed that Compound 1 did not induce apositive mutagenic response with strains TA98 and TA100 in presence andabsence of microsomal enzymes when the test articles were tested up tothe maximum concentration of 1000 μg/well (equivalent to 5000 μg/platein standard Ames assay).

TABLE 5 Mutagenicity Assay Results of Compound 1 REVERTANTS PER WELLBackground Lawn^(c) Conc. TA98 TA100 TA98/ μg/well 1 2 Mean 1 2 MeanTA100 MICROSOMES: NONE (−S9) DMSO — 5 6 6 26 32 29 4/4 Compound 1 62.5 44 4 34 30 32 4/4 Compound 1 125 4 6 5 32 34 33 4/4 Compound 1 250 6 5 634 29 32 4, sp/4, sp Compound 1 500 5 3 4 33 31 32 3, sp/3, sp Compound1 1000 4 5 5 28 26 27 3, mp/3, mp POSITIVE CONTROL^(a) 55 6359* >300 >300 >300*  4/4 MICROSOMES (+S9) DMSO — 6 6 6 34 33 34 4/4Compound 1 62.5 4 6 5 34 30 32 4/4 Compound 1 125 6 3 5 28 30 29 4/4Compound 1 250 8 6 7 30 28 29 4/4 Compound 1 500 6 6 6 32 37 35 3, sp/3,sp Compound 1 1000 6 7 7 33 34 34 3, mp/3, mp POSITIVECONTROL^(b) >300 >300 >300*  >300 >300 >300*  4/4 ^(a)TA98:2-nitrofluorene, 0.4 μg/well; TA100: Sodium azide, 2.0 μg/well ^(b)TA98and TA100: 2-aminoanthracene, 0.8 μg/well ^(c)Background Lawn EvaluationCodes: 5. enhanced growth compared to the solvent controls, 4. similaras vehicle control (normal, no toxicity), 3. less than 25% reduction(less than 25% cytotoxicity), 2. more than 25% but less than 50%reduction (less than 50% cytotoxicity), 1. more than 50% reduction (morethan 50% cytotoxicity), 0. no growth (100% cytotoxicity). sp = slightprecipitate mp = moderate precipitate hp = heavy precipitate *positiveincrease

Example 18 Pharmacodynamic Study in Tumor Cell Lines

Tumor cell lines H460 (Kras, PI3K), BT474 (HER2, PI3K), A375 (B-Raf) andH1975 (EGFR, PI3K) were cultured and treated with DMSO alone (vehiclecontrol) or 0.1 μmol/L Compound 1 or reference compound for 16 hours.Cell extracts were prepared in the presence of SDS and 2-mercaptoethanoland resolved in polyacrylamide gels. Proteins were transferred tonitrocellulose filter and blotting was done using standard procedureswith blocking solutions (Li-Cor Bioscience) containing the indicatedprimary antibody. Primary antibodies against p-EGFR, EGFR, p-HER2, HER2,p-HER3, HER3, p-MET, MET, p-bRaf, p-cRaf, pMEK, MEK, p-ERK, ERK andtubulin were purchased from Cell Signaling Technology. Secondaryantibody conjugated with IRdye 680, 800CW were used and the signal wasdetected with Li-Cor Odyssey Imager.

Immunocytochemistry was performed on cells grown in monolayer culturethat were treated as indicated in the figure legends and then fixed in4% (w/v) paraformaldehyde. After washing in 1×PBS, immunostaining wasperformed in Li-Cor blocking solution containing the indicated primaryantibodies and IRDye 680- or 800CW-conjugated secondary antibodies. Forin-cell-western, a Li-Cor Odyssey infrared imager was used for detectionand quantification of results.

For histological examination of pharmacodynamic markers, tumorxenografts were harvested and embedded in paraffin, and then 4-5-mmsections were prepared. The sections were mounted on slides and reactedwith primary antibodies followed by horseradish peroxidase-conjugatedsecondary antibody (Envision polymer-HRP, Dako, Glostrup, Denmark). Thecolor reaction was then performed using diaminobenzidine (DAB) asrecommended by the supplier. Counterstaining of the sections was donewith hematoxylin.

The results of this study are summarized in FIGS. 7A-7 g and 8A-8C.Compound 1 inhibits HDAC activity and PI3K pathway signaling in KRAS-and PI3KCA-mutant H460 non-small cell lung cancer (NSCLC) cells. Cellswere treated with DMSO alone (vehicle control) or containing testcompounds for 1 h before Western blot or in-cell-western was performed.FIG. 7A shows that Compound 1 at 1 μmol/L increases the levels ofacetylated histone 3 (Ac-H3), tubulin (Ac-Tub), and p53 (Ac-p53). Thecompound also upregulates total p53 and p21 content. The data set forthin FIGS. 7B-7E show that Compound 1 increases levels ofacetylated-tubulin (FIG. 7B), acetylated histone 3 (FIG. 7C), acetylatedp53 (FIG. 7D), and acetylated p21 (FIG. 7E) in a dose-dependent manner.Resulting IC₅₀ values suggest that Compound 1 has comparableHDAC-inhibitory potency to LBH 589 in the cancer cells examined. At 1μmol/L, Compound 1 inhibits the activation of AKT and the downstreamsignaling proteins 4EBP-1 and p70S6 (FIG. 7F). Compound 1 alsopersistently and potently inhibits phosphorylation of Akt in adose-dependent manner (FIG. 7G).

One major limitation of PI3K inhibitors in the treatment of cancers isthe activation of the RAF-MEK-ERK pathway. HDAC inhibitors are able toinhibit kinase levels in this signaling pathway in cancer cells viaepigenetic modification. In tumors cells with various mutations, such asthe KRAS and PI3K mutations in H460 cells, the B-Raf mutation in A375cells, HER2 and PI3K mutations in BT-474 cells, and EGFR mutations inH1975 cells, 100 nM Compound 1 suppressed activation of Raf, MEK, andERK. The potent HDAC inhibitor LBH 589 showed similar activities in someof these Western blot assays (FIG. 8A).

In addition to inhibition of the PI3K and MEK pathways, treattment ofRPMI-8226 myeloma cells with 1 μM Compound 1 for 16 h inhibited p-STAT3(Y-705) and p-Src (FIG. 8B).

In EGFR-L858R-T790M double-mutant H1975 NSCLC cells andHER2-overexpressing BT-474 breast cancer cells, Compound 1 was shown toreduce the levels of phosphorylated and total receptor tyrosine kinasesEGFR, HER2, HER3, and MET after incubation for 16 h. Similardownregulation of the same kinases was observed after treatment of thesecells with LBH 589 (FIG. 8C).

Example 19 Expression of PI3K110 α, β, γ and δ in HematologicalXenograft Tumor Models

Female immuno-deficient mice (Beige/SCID) at age 6-8 weeks were housedin ventilated micro-isolator cages in a controlled climate, fed withsterile high-fat diet (Problab-RMH 2000) ad libitum and providedsterilized water. All housing and supplies for SCID beige mice weredisposable, and purchased irradiated from Innovive prior to use. Micewere inspected daily including weekends/holidays by trained animalfacility personnel and investigators. All animal procedures wereperformed under sterile conditions within a biosafety cabinet (forinjections) or laminar flow hood (for animal husbandry and non-invasiveprocedures).

Human hematological cancer cell lines were originally obtained fromhuman cancer patients. Cryopreserved cells were thawed in a 37° C. waterbath and cultured in RPMI medium plus 10-15% Fetal Bovine Serum (FBS) ina tissue culture incubator at 5% CO₂. Cells were sent to outside vendorsfor contaminants and rodent pathogen screening intended to rule outcontamination by mycoplasma (by PCR) and/or virus (by MAP test, MouseAntibody Production).

When the cells in culture reached desired number, they were harvestedand washed with serum free Dulbecco's phosphate buffered saline (DPBS).Finally the cells were diluted in DPBS for implantation. Onlysingle-cell suspensions of greater than 90% viability (by trypan blueexclusion) were used for injection. After a seven day acclimationperiod, 10 to 20 million cells per animal suspended in 0.1 ml DPBS wereinjected subcutaneously (SC) in the right hind flank region of theanimal using a 0.5 CC syringe with a 26G hypodermic needle, taking careto avoid blood vessels. Successful implantation was indicated by theformation of a round, raised mass under the skin. The implanted micewere monitored for general health and tumor development daily.

Tumors were detectable about two and half weeks following implantation.Tumor size was measured with a caliper. The following formula was usedto calculate the tumor volume:

Tumor volume=(length×width²)/2

When tumor sizes reached about 150-300 mm³, mice were separated intofour groups including three treatment groups (25 mg/kg, 50 mg/kg and 100mg/kg) and one control group. Following dosing with Compound 1, tumorswere collected at 15 minutes, 1, 3, 6, 24 hours (3 mice for each timepoint). Tumors were collected according to the time points listed aboveafter mice were euthanized with CO₂. Samples were placed in the dry iceuntil transferred to a −80° C. freezer for Western blot analysis.

Protein was extracted from the tumor tissues using a homogenizer(Tissuelyser, Qiagen, Valencia, Calif.) according to the manufacturer'sinstructions. The adapters for holding the tissue tubes were frozen at−20° C., and the lysis buffer and beads were chilled at 4° C. beforeuse.

100-200 μg tissue was homogenized in 300 μl T-PER Mammalian TissueProtein Extraction reagent (Pierce, Rockford, Ill.) supplemented withphosphatase inhibitors (1:100 v/v, Tyr & Ser/Thr phosphatase inhibitorcocktails, Upstate). The specimen were checked visually after each cycle(time: 0.15 minutes; frequency: 30 Hz) until tissues were fullyhomogenized. Approximately four cycles were needed in most cases. Thetissue lysates were centrifuged at 14,000 rpm at 4° C. for 10 minutes.200 μl supernatant was collected and kept at −80° C. Proteinconcentration was measured using the BCA Protein Assay kit (Pierce,Rockford, Ill.) according to the manufacturer's instructions.

30 μg of total protein extract was resolved on NuPAGE Novex 4-12%Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes(Bio-Rad) using a Bio-Rad Semi-Dry Transfer Machine. The blots wereincubated with 10 ml Blocking Buffer (Odyssey Infrared Imaging System)for 1 hour and then probed with the primary antibody overnight at 4° C.on a shaker. The blots were probed with the primary antibody overnightat 4° C. Primary antibodies included PI3 Kinase p110α (#4249, 1:1000,Cell Signaling), PI3 Kinase p110β (#3011, 1:1000, Cell Signaling), PI3Kinase p110 γ (#5405, 1:1000, Cell Signaling), PI3 Kinase p110 δ(SC-7176 (1:1000, Santa Cruz Biotechnology, Santa Cruz, Calif.). GAPDH(glyceraldehyde 3-phosphate dehydrogenase, 1/30,000, Abcam, Cambridge,Mass.) was used as an internal control for each assay.

The membrane was rinsed four times with Tris-buffered saline Tween-20(TBST;DAKO) and incubated for 1 hour at room temperature with theinfrared conjugated secondary antibodies (1:10000): anti-Rabbitconjugated-IR Dye 800(Rockland), or anti-Mouse conjugated-Alexa680(Molecular Probes). The membrane was washed with TBST and then placedin the Odyssey Infrared Imaging System for imaging and analysis.

The results are set forth in FIG. 15, which shows Western blots of PI3Kp110 isoforms, AKT and pAKT from several Non-Hodgkin's Lymphoma andmultiple myeloma xenografts. The results show that activation of AKT isdriven by multiple PI3K P110 isoforms.

Example 20 Comparison of Compound 1 and CAL-101 in the Daudi XenograftTumor Model

Female SCID beige mice (CD-1 Beige SCID) at 6-8 weeks of age were housedin ventilated micro-isolator cages in a controlled climate, fed withsterile high-fat diet (Problab-RMH 2000 ad libitum and provided withsterilized water. All housing and supplies for SCID beige mice aredisposable, and purchased irradiated from Innovive prior to use. Micewere inspected daily including weekends/holidays by trained animalfacility personnel and investigators. All animal procedures wereperformed under sterile conditions in a biosafety cabinet (forinjections) or laminar flow hood (for animal husbandry and non-invasiveprocedures).

Daudi human Burkitt's lymphoma cells were originally obtained from ahuman Burkitt's lymphoma patient. Cryopreserved cells were thawed in a37° C. water bath and cultured in RPMI-1640 medium plus 15% Fetal BovineSerum (FBS), 1% Penstrep, and 1% Glutamax in a tissue culture incubatorat 5% CO₂. Cells were sent to outside vendors for pathogen screeningintended to rule out contamination by mycoplasma (by PCR) and/or virus(by MAP test, Mouse Antibody Production). When the cells in culturereached desired numbers, they were harvested by centrifuging. Aftercollection, the cells were washed with serum-free Dulbecco's phosphatebuffered saline (DPBS). Finally the cells were diluted in DPBS forimplantation. Only single-cell suspensions of greater than 90% viability(by trypan blue exclusion) were used for injection and 20 million cellsper animal suspended in 0.1 ml DPBS were injected subcutaneously in theright hind flank region of the mouse after a minimum 7 day acclimationperiod, using a 0.5 CC syringe with a 26G hypodermic needle, taking careto avoid blood vessels. Successful implantation was indicated by theformation of a round, raised mass under the skin. The implanted micewere monitored for general health and tumor development daily.

Tumors were detectable about two weeks following implantation. Tumorsize was measured with a caliper. The following formula was used tocalculate the tumor volume:

Tumor volume=(length×width²)/2

Four weeks after tumor implantation, tumors reached an average size of300±126 mm³. Animals with acceptable tumor size and shape were randomlyassigned into three groups of seven animals each, using sortingsoftware, one vehicle control and two treatment groups.

Number Dose Groups of mice Compounds (mg/kg) Schedule 1 7 30% Captisol 0Qd* (Mon-Fri), PO** 2 7 CAL-101 30 BID*** (Mon-Fri) 3 7 Compound 1 100Qd* (Mon-Fri), PO** *Qd = Once daily dosing, **PO = Oral Gavage dosing,***BID, twice daily

Compound 1 was formulated and dosed as follows: Compound 1 (7.5 mg/ml)was dissolved in 30% Captisol with 2 molar equivalents of NaOH, balancedwith 2 molar equivalents of HCl, and dosed via oral gavage daily Mondaythrough Friday. The control group was dosed with vehicle (30% Captisol)using the same dosing paradigm as the 100 mg/kg volume (6.67 ul/g).

During each animal study, tumors were measured with calipers, tumor sizedetermined using the above mentioned formula, and tumor size changes inpercentage calculated. Mouse body weights were measured with a scaletwice per week. Studies were continued until either: a) thepredetermined end date indicated in the study design; or b) the onset ofhealth problems, whichever occurred first. In addition, the followingtumor-related parameters warranted provision of euthanasia: tumor burdenexceeding 2500 mm³ and/or loss of ≧20% of starting body weight. Inaddition to the determination of tumor size changes, the last tumormeasurement was used to generate the tumor weight change ratio (T/Cvalue), a standard metric developed by the National Cancer Institute(NCI) for xenograft tumor evaluation. T/C values were calculated usingthe following formula: % T/C=100×ΔT/ΔC if ΔT>0. In cases where tumorregression occurred, however, the following formula was used: %T/T₀=100×ΔT/T0 if ΔT<0.

The treatment period was 15 days for the vehicle and CAL-101 groups,which required earlier termination due to tumor size exceeding 10% ofbody weight, and 18 days for the Compound 1 group. Tumor sizes and bodyweights were measured again on the last day of the study.

The results of the study are presented in FIG. 16, which shows tumorgrowth for the active and control groups as a function of treatmenttime. The Compound 1 group showed significantly reduced tumor growthcompared to the CAL-101 and control groups.

Example 21 Combination of Compound 1 and Cyclophosphamide in the DaudiXenograft Tumor Model

Female SCID beige mice (CD-1 Beige SCID) at 6-8 weeks of age were housedin ventilated micro-isolator cages in a controlled climate, fed withsterile high-fat diet (Problab-RMH 2000 ad libitum and provided withsterilized water. All housing and supplies for SCID beige mice aredisposable, and purchased irradiated from Innovive prior to use. Micewere inspected daily including weekends/holidays by trained animalfacility personnel and investigators. All animal procedures wereperformed under sterile conditions in a biosafety cabinet (forinjections) or laminar flow hood (for animal husbandry and non-invasiveprocedures).

Daudi human Burkitt's lymphoma cells were originally obtained from ahuman Burkitt's lymphoma patient. Cryopreserved cells were thawed in a37° C. water bath and cultured in RPMI-1640 medium plus 15% Fetal BovineSerum (FBS), 1% Penstrep, and 1% Glutamax in a tissue culture incubatorat 5% CO₂. Cells were sent to outside vendors for pathogen screeningintended to rule out contamination by mycoplasma (by PCR) and/or virus(by MAP test, Mouse Antibody Production). When the cells in culturereached desired numbers, they were harvested by centrifuging. Aftercollection, the cells were washed with serum-free Dulbecco's phosphatebuffered saline (DPBS). Finally, the cells were diluted in

DPBS for implantation. Only single-cell suspensions of greater than 90%viability (by trypan blue exclusion) were used for injection and 20million cells per animal suspended in 0.1 ml DPBS were injectedsubcutaneously into the right hind flank region of the mouse after aminimum 7 day acclimation period, using a 0.5 cc syringe with a 26Ghypodermic needle, taking care to avoid blood vessels. Successfulimplantation was indicated by the formation of a round, raised massunder the skin. The implanted mice were monitored for general health andtumor development daily.

Tumors were detectable about two weeks following implantation. Tumorsize was measured with a caliper. The following formula was used tocalculate the tumor volume:

Tumor volume=(length×width²)/2

Four weeks after tumor implantation, tumors reached an average size of189±47 mm³. Animals with acceptable tumor size and shape were randomlyassigned into four groups of eight animals each, using sorting software,one vehicle control and three treatment groups.

Dose Groups Number of mice Compounds (mg/kg) Schedule 1 8 30% Captisol 0Qd* (Mon-Fri), 0.9% NS PO** 2 8 Compound 1 75 Qd* (Mon-Fri), PO** 3 8CTX 50 Day-0, iv 4 8 Compound 1 75 Qd* (Mon-Fri), +CTX 50 PO** Day-0, iv*Qd = Once daily dosing, **PO = Oral Gavage dosing, ***BID, twice daily

Compound 1 was formulated and dosed as follows: Compound 1 (7.5 mg/ml)was dissolved in 30% Captisol with 2 molar equivalents of NaOH, balancedwith 2 molar equivalents of HC1, and dosed via oral gavage daily Mondaythrough Friday at 75mg/kg.

Cyclophosphamide (“CTX”) was dissolved in 0.9% NS at 5mg/ml, and dosediv (tail vein injection) to animals at 50mg/kg on Day-0. The combinationgroup was dosed with both Compound 1 and CTX using same dosing schedule.The control group was dosed with vehicle (30% Captisol) and 0.9% NSusing the same paradigm as for the combination.

During each animal study, tumors were measured with calipers, tumor sizedetermined using the above mentioned formula, and tumor size changes inpercentage calculated. Mouse body weights were measured with a scaletwice per week. Studies were continued until either: a) thepredetermined end date indicated in the study design; or b) the onset ofhealth problems, whichever occurred first. In addition, the followingtumor-related parameters warranted provision of euthanasia: tumor burdenexceeding 2500 mm³ and/or loss of 20% of starting body weight. Inaddition to the determination of tumor size changes, the last tumormeasurement was used to generate the tumor weight change ratio (T/Cvalue), a standard metric developed by the National Cancer Institute(NCI) for xenograft tumor evaluation. T/C values were calculated usingthe following formula: % T/C=100×ΔT/ΔC if ΔT>0. In cases where tumorregression occurred, however, the following formula was used: %T/T₀=100×ΔT/T0 if ΔT<0.

The treatment period was 2 weeks. Tumor sizes and body weights weremeasured again on the last day of the study.

The results of this study are set forth in FIG. 17, which shows tumorgrowth as a function of treatment time for the control and treatmentgroups. As single agents, Compound 1 and cyclophosphamide ave similaractivity in this model. The combination of Compound 1 andcyclophosphamide showed substantially greater effeicacy than either agenalone.

Example 22 Compound 1 in Combination with Lenalidomide in the MM1SXenograft Model

Female SCID/Beige mice at age 4 weeks were housed in ventilatedmicro-isolator cages (INNOCAGE®IVC, Innovive Inc., San Diego, Calif.) ina controlled climate, fed with sterile high-fat diet (Problab-RMH 2000)ad libitum and provided with sterilized water. All housing and suppliesfor SCID/Beige mice were sterilized by autoclaving before use. Mice wereinspected daily including weekends/holidays by trained animal facilitypersonnel and investigators. All animal procedures were performed understerile conditions within a biosafety cabinet (for injections) orlaminar flow hood (for animal husbandry and non-invasive procedures).

Cryopreserved MM1S human MM cells were thawed in a 37° C. water bath andcultured in RPMI medium plus 10% Fetal Bovine Serum (FBS) in a tissueculture incubator at 5% CO₂. Cells were sent to outside vendors forcontaminants and rodent pathogen screening intended to rule outcontamination by mycoplasma (by PCR) and/or virus (by MAP test, MouseAntibody Production). When the cells in culture were enough forimplantation, they washed with serum free Hank's balanced salt solution(HBSS). Finally the cells were diluted in HBSS for implantation. Onlysingle-cell suspensions of greater than 90% viability (by trypan blueexclusion) were used for injection and 20 million cells per animalsuspended in 0.2 ml HBSS were injected subcutaneously in the right hindflank region of the mouse after a minimum 7 day acclimation period,using a 1 CC syringe with a 26G hypodermic needle, taking care to avoidblood vessels. Successful implantation was indicated by the formation ofa round, raised mass under the skin. The implanted mice were monitoredfor general health and tumor development daily.

Tumors were detectable about two weeks following implantation. Tumorsize was measured with a caliper. The following formula was used tocalculate the tumor volume:

Tumor volume=(length×width²)/2

Three weeks after tumor implantation, tumor reached an average of 192±32mm³. Animals with acceptable tumor size and shape were randomly assignedinto 6 groups of 7 animals each, using sorting software, one vehiclecontrol and six treatment groups.

Number Dose Groups of mice Compounds (mg/kg) Schedule 1 7 30% Captisol 0Qd* (Mon-Fri), PO** MCT Qd* (Mon-Fri), PO** 2 7 Compound 1 75 Qd*(Mon-Fri), PO** 3 7 Lenalidomide 12.5 Qd* (Mon-Fri), PO** 4 7Lenalidomide 12.5 Qd* (Mon-Fri), PO** 5 7 Compound 1 75 Qd* (Mon-Fri),PO** +Lenalidomide 12.5 Qd* (Mon-Fri), PO** 6 7 Compound 1 75 Qd*(Mon-Fri), PO** +Lenalidomide 25 Qd* (Mon-Fri), PO**

Compound 1 was formulated and dosed as follows: Compound 1 (7.5 mg/ml)was dissolved in 30% Captisol with 2 molar equivalents of NaOH, balancedwith 2 molar equivalents of HCl, and dosed via oral gavage daily Mondaythrough Friday at 75mg/kg. Lenalidomide (Selleck, 2.5mg/m1) wasformulated in MCT (0.5% methyl cellulose and 0.2% Tween80), and dosed at12.5 mg/kg or 25mg/kg. The two combinations groups were dosed withCompound 1 at 75 mg/kg plus one dose level of lenalidomide (either 12.5or 25 mg/kg). The control group was dosed with vehicle (30% Captisol)and MCT using the same paradigm as for the combination.

During each animal study, tumors were measured with calipers, tumor sizedetermined using the above mentioned formula, and tumor size changes inpercentage calculated. Mouse body weights were measured with a scaletwice per week. Studies were continued until either: a) thepredetermined end date indicated in the study design; or b) the onset ofhealth problems, whichever occurred first. In addition, the followingtumor-related parameters warranted provision of euthanasia: (1) tumorburden exceeding 2500 mm³ and/or (2) loss of 20% of starting bodyweight. In addition to the determination of tumor size changes, the lasttumor measurement was used to generate the tumor weight change ratio(T/C value), a standard metric developed by the National CancerInstitute (NCI) for xenograft tumor evaluation. T/C values werecalculated using the following formula: % T/C=100×ΔT/ΔC if ΔT>0. Incases where tumor regression occurred, however, the following formulawas used: % T/T₀=100×ΔT/T0 if ΔT<0.

The treatment period was 17 days. Tumor sizes and body weights weremeasured again on the last day of the study.

The results of this study are presented in FIG. 18, which shows tumorgrowth as a function of treatment time. The results show that Compound 1at 75 mg/Kg PO is more effective than Lenalidomide at either 12.5 or 25mg/Kg PO as single agents. The results also show that the combination ofCompound 1 and lenalidomide is significantly more effective than eithercompound alone.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein R is hydrogen oran acyl group.
 2. The compound of claim 1 wherein R is R₁C(O)—, whereinR₁ is substituted or unsubstituted C₁-C₂₄-alkyl; substituted orunsubstituted C₂-C₂₄-alkenyl, preferably C₂-C₁₀-alkenyl, and morepreferably C₂-C₆-alkenyl; substituted or unsubstituted C₂-C₂₄-alkynyl;substituted or unsubstituted aryl; or substituted or unsubstitutedheteroaryl.
 3. The compound of claim 1 wherein R is H or acetyl.
 4. Thecompound of claim 1 represented by the formula:

or a pharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising as an active ingredient a compound of claim 1 anda pharmaceutically acceptable carrier.
 6. A pharmaceutical compositionfor oral administration comprising as an active ingredient a compound ofclaim 1 and a pharmaceutically acceptable carrier.
 7. A pharmaceuticalcomposition comprising as an active ingredient a compound of claim 4 anda pharmaceutically acceptable carrier.
 8. A pharmaceutical compositionfor oral administration comprising as an active ingredient a compound ofclaim 4 and a pharmaceutically acceptable carrier.
 9. A method oftreating a PI3K related disease or disorder in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of the pharmaceutical composition ofclaim
 5. 10. The method of claim 9, wherein said PI3K related disease ordisorder is a cell proliferative disorder.
 11. The method of claim 10,wherein the cell proliferative disorder is a cancer.
 12. The method ofclaim 11, wherein the cancer is selected from the group consisting ofpapilloma, blastoglioma, Kaposi's sarcoma, melanoma, non-small cell lungcancer, ovarian cancer, prostate cancer, squamous cell carcinoma,astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer,lung cancer, colorectal cancer, thyroid cancer, pancreatic cancer,gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin'sdisease and Burkitt's disease.
 13. A method of treating an HDAC-mediateddisease comprising administering to a subject in need thereof apharmaceutical composition of claim
 5. 14. A method of treating bothPI3K and HDAC mediated diseases comprising administering to a subject inneed thereof a pharmaceutical composition of claim
 5. 15. A method oftreating a PI3K related disease or disorder in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of the pharmaceutical composition ofclaim
 7. 16. The method of claim 13, wherein said PI3K related diseaseor disorder is a cell proliferative disorder.
 17. The method of claim16, wherein the cell proliferative disorder is a cancer.
 18. The methodof claim 17, wherein the cancer is selected from the group consisting ofpapilloma, blastoglioma, Kaposi's sarcoma, melanoma, non-small cell lungcancer, ovarian cancer, prostate cancer, squamous cell carcinoma,astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer,lung cancer, colorectal cancer, thyroid cancer, pancreatic cancer,gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin'sdisease and Burkitt's disease.
 19. A method of treating an HDAC-mediateddisease comprising administering to a subject in need thereof apharmaceutical composition of claim
 7. 20. A method of treating bothPI3K and HDAC mediated diseases comprising administering to a subject inneed thereof a pharmaceutical composition of claim 7.